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High-Throughput Real-Time Quantitative Reverse

1. Primer mixes 1 25 uM each forward and reverse primer Mix small aliquots of 2 5 uM forward and reverse primer stocks see recipe in equal volumes 1 1 Store up to 1 to 2 months at 4 C in screw capped tubes to prevent evaporation Do not freeze as repeated freeze thaw cycles will degrade the primers Primer stocks 100 and 2 5 uM Purchase lyophilized oligonucleotides from any commercial source synthesized at a 25 nM scale standard desalting is sufficient no additional purification is needed Briefly centrifuge the tubes of powdered oligonucleotide to get contents to the bottom Resuspend in molecular biology grade water to 100 uM Before preparing primer mixes dilute each stock forward and reverse to 2 5 uM Store at 20 C When stored properly and subjected to minimal freeze thaw cycles primer stocks can last gt 2 years Primer pairs produced at a 25 nM scale will yield enough reagent to test 4000 samples using SYBR Green or 2000 samples using TaqMan assays Sterile filtered double deionized water may be used instead of purchased molecular grade water Do not use DEPC treated water because the slightly acidic pH may promote primer degradation Tris buffer may be used instead of water but should not contain EDTA which acts as a Mg chelating agent and can inhibit the PCR Standard cDNAs dilution series DNase treat UNIT 3 12 and reverse transcribe UNIT 3 7 a suitable RNA using 0 08 ug l random hexamer primers such
2. Contributed by Angie L Bookout Carolyn L Cummins and David J Mangelsdorf Howard Hughes Medical Institute University of Texas Southwestern Medical Center Dallas Texas Jean M Pesola and Martha F Kramer preparation of RNA standards Harvard Medical School Boston Massachusetts Current Protocols in Molecular Biology
3. Several vendors e g Ambion Clontech Stratagene supply total RNA preparations of many cell and tissue types collected from many different species Both Stratagene and Clontech make total RNA pools termed universal reference RNA composed of mixtures of either vari ous cell lines or whole tissues respectively These RNA pools represent gt 90 gene cov erage on microarrays Novoradovskaya et al 2000 Clontech 2002 and are very useful as standards both for validation of primer probe sets and for large scale multigene studies Current Protocols in Molecular Biology Absolute RNA standards The absolute quantification of RNA syn thetic standards by radiolabel incorporation permits accurate determination of transcript amounts Liquid scintillation counting of the filters typically yields incorporation values with errors of less than 5 However er rors of up to 10 to 15 will not significantly affect quantification in most applications The use of low energy S compared to P at very low specific activity levels minimizes po tential radiolytic degradation of the synthetic RNA over time and minimizes personal radia tion exposure Very small amounts of standard RNA are typically needed for an RT PCR as say less than 108 copies such that the amount of 35S in each RT PCR reaction will be ex tremely small One consideration is the purity of the radio labeled ribonucleotide Fresh lots are generally guarantee
4. F 2002 Accurate normalization of real time quan titative RT PCR data by geometric averaging of multiple internal control genes Genome Biol 3 1 12 Whitcombe D Theaker J Guy S P Brown T and Little S 1999 Detection of PCR products using self probing amplicons and fluorescence Nat Biotechnol 1717 804 807 Whittwer C T Herrmann M G Moss A A and Rasmussen R P 1997 Continuous fluorescence monitoring of rapid cycle DNA amplification BioTechniques 22 130 138 Key References Ambion 2001 The top 10 most common quantita tive RT PCR pitfalls Technotes Newsletter 8 8 Ambion Houston Tex A short but useful checklist of critical considera tions for performing any type of reverse transcrip tion PCR Applied Biosystems 1997 Relative quantitation of gene expression ABI PRISM 7700 Sequence Detection System User Bulletin 2 Rev B Ap plied Biosystems Foster City Calif This bulletin outlines both the standard curve and AACt methods and shows by comparing data ob tained using both calculations that the resulting values are very similar regardless of the assay Applied Biosytems 2001a See above This instrument manual contains explanations about the transformation of fluorescence signal into Ct data in addition to outlining the proper method of baseline and threshold settings for ABI machines Users should consult their specific instrument man uals since each type of instrumentation
5. input cpm x initial reaction volume at step 6 x NTP purity This calculation assumes a uniform product length The NTP purity term refers to the fraction of S present in intact NTP molecules This amount is typically 0 90 to 0 99 in fresh radiochemical preparations See Commentary for more details At least 30 of radiolabel should be incorporated for simple templates and at least 10 for difficult templates with a high degree of secondary structure Multiply this fraction by the total amount nmol of radiolabeled plus unlabeled CTP in the reaction to obtain the amount in nmol of cytidine incorporated into product RNA Divide by the fraction of cytidine residues in the transcript to calculate the total nmol of ribonucleotides incorporated into product RNA Multiply this value by 10 mol nmol and by Avogadro s number 6 022 x 1073 molecules mol to obtain the total number of incorporated ribonucleotides Divide by the transcript length to determine the number of transcripts composing the purified RNA Make duplicate serial dilutions of one or more transcripts quantified in this manner and process them through the DNase treatments and reverse transcription in parallel with the experimental RNA samples By using duplicate standards one can ensure that the protocols used to prepare cDNA are quantitative and reproducible Five or six serial 10 fold dilutions are usually suitable Exact quantities depend upon the anticipated range of ex
6. Basic Protocol 1 is the most basic and straightforward QPCR assay described in the unit In this method standard curves are constructed for all of the GOIs from which RNA expression is being measured and linear regression analysis is applied to interpolate unknown sample values The standard curve assay may be performed even if the PCR amplification efficiencies of the primer sets as determined by the template dilution assay in Support Protocol 2 are not equal since correction for unequal efficiencies is intrinsic to the linear regression formula One drawback of the standard curve method is that standard curves must be run for each of the primer sets on an assay plate This results in less space on the plate for the unknown samples and requires the use of additional reagents This use of resources is particularly excessive when the PCR amplification efficiencies of the primer sets have been determined to be essentially 100 and relative fold change is the preferred outcome of the measurements In such cases the AACt method see Alternate Protocol should be employed instead Another limitation is that unless the levels of all of the GOIs in the cDNAs used to construct the standard curves are known the relative concentration of one GOI cannot be compared to that of another GOI If comparison between the levels of different GOIs without the knowledge of the relative level of the transcripts in the standards is desired the efficiency corrected ACt
7. RNA in an unknown sample can be calculated from its Ct DESIGN AND VALIDATION OF SYBR GREEN AND TaqMan PRIMER PROBE SETS Considering that QPCR relies on the quality and the fidelity of the primers and probes that are used very strict parameters for their design and subsequent validation are required A common misconception in performing QPCR assays is that if the primer works for traditional end point PCR it is suitable for QPCR In some cases this is true However the primer set must be tested in a QPCR validation assay before it can be used for RNA expression analysis In keeping with the high throughput capacity of QPCR the thermocycling conditions are kept constant for all assays 10 min at 95 C activates the hot start Taq polymerase followed by 40 cycles of the two step 95 C melting and 60 C annealing An extension step in the thermocycler program is not required since all of the PCR products are 50 to 150 bp thus making the run last only 1 5 to 2 hr The concentrations of PCR reagents such as Tag DNA polymerase MgClo other salts and dNTPs remain constant within the same chemistry i e SYBR Green or TaqMan and these so called universal cycling conditions make primer and probe sequences the only point of flexibility in performing the assays The design of primer probe sets requires the availability of reliable sequence informa tion that may be obtained from databases like NCBI s GenBank or Ensembl or from data produced by dir
8. The instrument software uses Current Protocols in Molecular Biology an algorithm to transform and display the melting curve as the negative first derivative of the normalized fluorescence versus temperature Applied Biosystems 2001a The presence of a single peak in the melting curve is indicative of a single PCR product and occurs at the melting temperature of the product Multiple peaks in this plot indicate that nonspecific products or primer dimers have been formed Formation of a single product can be confirmed by running the PCR products on a 2 agarose or 10 polyacrylamide gel following the QPCR run Occasionally when two products are observed the second product may have been formed during the plateau phase which would not affect quan tification To confirm whether this has occurred the QPCR run could be repeated and stopped during the exponential phase and the reaction products run on an agarose gel However this is not feasible in practice because of the high throughput nature of the assay The best course of action when multiple products are observed in the dissociation curve is to redesign and validate a new primer set Only primer sets that give a single peak in this curve should be used for experimental assays Once a SYBR Green based primer set has passed validation testing the corresponding TaqMan probe is ordered and validated for PCR efficiency only In rare cases the SYBR Green assay conditions e g primer concentratio
9. chloroform isoamy alcohol and finally ethanol precipita tion UNIT 2 1A in the presence of sodium acetate Resuspend DNA in nuclease free water Acceptable results may also be obtained by using an enzymatic reaction clean up kit such as the QIAquick PCR Purification Kit Qiagen or by performing gel extraction UNIT 2 6 If RNase contamination is a problem the sample can be treated with 50 to 100 jxg ml proteinase K to destroy the RNases prior to purification The concentration of linearized template does not need to be determined The authors assume that most of the 10 ug used in the digest are recovered Synthesize RNA 4 For each transcription reaction prepare four 1 5 ml screw cap microcentrifuge tubes each containing 24 ul of 0 5 ug ml sheared salmon sperm DNA one pair to measure Current Protocols in Molecular Biology 10 11 12 13 14 input 35S in duplicate and one pair to measure 35S incorporation in duplicate Also prepare one single tube as a control with no other additions Set these tubes aside on ice The salmon sperm DNA will act as a carrier for the synthesized RNA during the acid precipitation step In another 1 5 ml screw cap microcentrifuge tube set up a 100 ul transcription reaction minus the enzyme as follows 40 mM Tris Cl pH 7 5 10 mM MgCl 5 mM DTT 500 uM 4NTP mix 1 ul 10 mCi ml 600 Ci mmol gt S CTP 2 to 5 ug DNA template from step 3 5 ug BSA 1 mM spermidin
10. cloned cDNA will produce sense transcripts 4 Engineer the plasmid such that cleavage by arestriction enzyme generates a linear piece of DNA that contains the phage polymerase promoter and the entire sequence of the desired synthetic RNA This is ideally accomplished using an enzyme which linearizes the plasmid by cutting at only one site at the end of the de sired transcript sequence Avoid including su perfluous vector sequences in the transcribed region since these will not be present in RNA from the experimental samples Avoid using restriction enzymes that cre ate 3 overhangs because there is evidence that they cause RNA complementary to the in tended transcripts to be generated Schenborn and Mierendorf 1985 As an alternative to a plasmid PCR prod ucts can also be designed for use as tran scription templates by incorporating the poly merase promoter sequence into one of the primers Mullis and Faloona 1987 Anticipated Results The setup of the AACt assay allows 64 samples one of which should be the NTC to be assayed for an endogenous reference gene and one gene of interest in triplicate the stan dard curve and efficiency corrected ACt as says will accommodate an endogenous refer ence gene and one gene of interest in triplicate for 56 unknowns six standards and two con trol samples Assays performed on the ABI 7900HT are completed in 1 5 and 2 hr for Taq Man and SYBR Green based assays respec tivel
11. cold 100 methanol ice cold Universal scintillation cocktail preferably biodegradable e g Ecoscint A National Diagnostics 1 5 ml screw cap microcentrifuge tubes DE81 paper Whatman Filter paper Current Protocols in Molecular Biology SUPPORT PROTOCOL 1 The Polymerase Chain Reaction es 15 8 11 Supplement 74 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 12 Supplement 74 Glass fiber filters Whatman GF C 24 mm discs Transparent plastic wrap Forceps Heat lamp optional 250 ml glass or metal beaker Liquid scintillation counter and vials Additional reagents and equipment for subcloning UNIT 3 16 plasmid minipreps UNIT 1 6 digestion with restriction endonucleases UNIT 3 1 agarose gel electrophoresis UNIT 2 54 purification of DNA UNIT 2 14 phage RNA polymerase reactions UNITS 3 4 amp 3 8 agarose formaldehyde or glyoxal gel electrophoresis UNIT 4 9 and drying and imaging of gels APPENDIX 3A CAUTION To minimize the risk of radioactive contamination use screw cap microcen trifuge tubes with cap gaskets and filtered pipet tips for all manipulations of radioactive solutions Wear gloves and dispose of all gt S contaminated material properly See AP PENDIX IF for more details on handling radioactivity CAUTION Phenol chloroform and trichloroacetic acid are hazardous see APPENDIX 1H NOTE For all procedures involving RNA use reagents and solution
12. if the desired output is only fold change see Basic Protocol 1 or Alter nate Protocol Data obtained from the efficiency corrected ACt method are expressed as normalized RNA level in arbitrary units and the calculated levels may be compared to those of other GOIs when the same threshold setting and assay chemistry are used i e SYBR Green or TaqMan chemistry It should be noted that an assumption is made that the reverse transcription efficiency is equal for all RNA transcripts in a single sample Current Protocols in Molecular Biology BASIC PROTOCOL 2 The Polymerase Chain Reaction 15 8 7 Supplement 73 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 8 Supplement 73 and for the same transcript between samples In some cases this may not be true see Pfaffl 2004 for further discussion therefore it is recommended that the RNA extrac tion method remain the same for all samples and that all samples under study be reverse transcribed at the same time with the same reaction buffer The basis of quantitative PCR lies in the principle that for every additional thermocycle a two fold increase of template specific product occurs Several factors affect whether a change in one cycle truly represents a two fold growth in product in other words whether the reaction is 100 efficient To assess the reaction s efficiency linear regression analysis is applied to a standard cDNA dilution s
13. primers Should have the same properties as the primers except that the melting temperature should be around 70 C and the sequence should not contain G s within a few bases of the 5 end because of increased reporter quenching In addition the sequence must have more C s than G s which can be accomplished by using the complementary strand sequence for the probe Applied Biosystems 2002b Primer Express contains templates into which these parameters have been preloaded Current Protocols in Molecular Biology The Polymerase Chain Reaction 15 8 17 Supplement 73 5 Choose a primer probe set for which the primers anneal in different exons or which have less than 5 bp overhangs into the adjacent exon on the 3 end of the primer This step is necessary to avoid amplification of contaminating genomic DNA Although the RNA is DNase treated prior to reverse transcription complete removal of genomic DNA is never achieved For intron less transcripts or other primer sets that bind sequence within a single intron the RT controls are essential for each sample to ensure that genomic DNA is not being amplified Perform a BLAST or equivalent search of both primers of the set together to verify that they will fully anneal to the correct sequence and only that sequence If the TaqMan probe will be used run BLAST um 19 3 on the probe sequence to assess whether it binds to the correct sequence with 100 ident
14. small genomic DNA may still be am plified This can be avoided by allowing a few base pairs on the 3 end of either primer to overhang onto the neighboring exon To distinguish knockout or mutant samples using QPCR primers probes should be de signed in the knocked out or mutated region of the transcript In this way no amplification of the transcript will occur for the knockout since the region recognized by the primers has been deleted or altered Primers for transcripts that are rapidly degraded should be placed near the 5 end of the RNA sequence as degrada tion by ribonucleases generally occurs 3 to Current Protocols in Molecular Biology 5 Brown 2002 However RNA transcripts that have undergone linear RNA amplification for instance RNA isolated from laser capture microdissected cells are around 200 to 1000 bases in length and represent the 3 ends of the transcripts In this case primers must be designed in a region near the poly A tail It is absolutely mandatory to validate both primers and probes on the same instrument used to perform the experimental assay i e when the instrument used to validate the primer sets is a different brand than the one that will be used for the experimental assays e g Roche iCycler versus ABI 7900HT Published primer probe sets for which the PCR product is lt 150 base pairs and the annealing temperature is around 60 C which have been validated properly should be
15. the following components as indicated in Table 15 8 4 25 mM MgCh molecular biology grade store at 20 C 10x Gold PCR buffer supplied with PCR enzyme Applied Biosystems store at 20 C 10x dNTP mix equal volumes of 2 mM dATP dTTP dCTP and dGTP store at 20 C Dimethylsulfoxide DMSO molecular biology grade store at room temperature SYBR Green I dye Molecular Probes store at 20 C diluted 1 1000 in water 50x ROX passive reference dye Invitrogen store at 20 C AmpliTaq Gold polymerase 5 U l Applied Biosystems store at 20 C Water molecular biology grade Prepare fresh and keep on ice prior to use in primer master mixes Protect dyes and all prepared mixes from prolonged exposure to light by wrapping tubes in foil To maintain the high throughput nature of the assay all buffer conditions including the concentrations of Mg dNTPs and other additives are kept constant It is strongly recommended that a preformulated buffer be purchased from a reliable vendor since these are stable at room temperature and have been quality control tested to ensure optimal performance The authors have obtained comparable results using mixes from Applied Biosystems Bio Rad Invitrogen and Sigma However it is important to compare lots as there can be lot to lot differences in the commercial preparations TaqMan probe 100 and 5 pM Depending on the vendor the probe may be supplied in a lyophilized form In
16. 1 Label glass fiber filters by cutting varied notches along the perimeter and lay them out on a sheet of plastic wrap with forceps Prepare four filters for the single tube of control salmon DNA step 4 four filters for each of the tubes of input S step 6 and two filters for each of the tubes of incorporated step 12 For the control salmon DNA and the input S samples two of each set of four replicate filters will be subjected to TCA precipitation and washing while the other two will be put directly into scintillation vials as unwashed samples For the incorporated 35S samples the two replicate filters will be subjected to TCA precipitation and washing Mix the tubes of salmon sperm DNA and spot 5 ul of each mixture onto the corre sponding two or four replicate filters Allow the spotted samples to air dry or use a heat lamp Use forceps to place duplicate filters from the control salmon DNA and the input S samples in individual scintillation vials and set aside as unwashed samples Place the remaining duplicate filters from the control salmon DNA and the input S samples along with the duplicate filters from the incorporated S samples into a 250 ml glass or metal beaker with 50 ml of 10 ice cold TCA This will precipitate the nucleic acids and wash away unincorporated rNTPs Up to 18 filters can be washed with 50 ml for more filters scale up the volumes proportionally Current Protocols in Molecular Bio
17. D Lewis J Raff M Roberts K and Watson J D 1994 The cell nucleus Jn Molecular Biology of the Cell 3 ed p 370 Garland Publishing New York Applied Biosytems 2001a ABI Prism 7900HT User Manual rev 4 Applied Biosystems Foster City Calif Current Protocols in Molecular Biology Applied Biosystems 2001b TaqMan human endogenous control plate Protocol Revision C http docs appliedbiosystems com pebiodocs 04308134 pdf Applied Biosystems Foster City Calif Applied Biosystems 2002a Real time PCR vs tra ditional PCR https www appliedbiosystems com support tutorials pdfirtpcr_vs_tradpcr pdf Applied Biosystems Foster City Calif Applied Biosystems 2002b Designing Taq Man MGB probe and primer sets for gene expression using Primer Express software http www appliedbiosystems com support tutorials pdfltaqman_mgb_primersprobes for gene_expression pdf Applied Biosystems Foster City Calif Applied Biosystems 2003 Creating standard curves with genomic DNA or plasmid DNA templates for use in quantitative PCR https www appliedbiosystems com support tutorials pdf quant_pcr pdf Applied Biosys tems Foster City Calif Brown T A 2002 How genomes function Jn Genomes 2nd ed S Carlson ed section 10 4 John Wiley amp Sons Hoboken N J Clontech 2002 Control RNA for microar ray experiments Clontechniques XVIL6 http www clontech com clontech archive APRO2UPD pdfiControl
18. High Throughput Real Time Quantitative Reverse Transcription PCR This unit describes the use of real time quantitative PCR QPCR for high throughput analysis of RNA expression The topics covered include the standard curve method see Basic Protocol 1 production and quantification of RNA standards see Support Protocol 1 an efficiency corrected ACt cycle time also called cycle threshold or crossing point method see Basic Protocol 2 the comparative cycle time or AACt method see Alternate Protocol and design and validation of QPCR primers and probes for both SYBR Green and TaqMan based assays see Support Protocol 2 While the unit describes the use of the Applied Biosystems 7900HT high throughput 384 well instrument the protocols may be utilized for any real time PCR instrument The high throughput design allows analysis of the levels of transcripts from a number of genes of interest GOIs at one time by using the appropriate primer set for each gene Within this unit the term GOI will refer to the actual gene of interest as well as its RNA product or cDNA copy Absolute quantification means that the absolute copy number of the GOI is measured Relative quantification means that a quantitative difference in copy number between two samples experimental and control is measured by normalizing both samples to an endogenous reference Because of the simplicity of the mathematical application the relative standard curve method
19. RNA pdf Clontech Palo Alto Calif Dheda K Huggett J F Bustin S A Johnson M A Rook G and Zumla A 2004 Vali dation of housekeeping genes for normalizing RNA expression in real time PCR BioTech niques 37 112 119 Guo D Henriksson R and Hedman H 2001 The iCycler iQ detection system for evaluating ref erence gene expression in normal human tissue rev A Amplification 2804 http www bio rad com LifeScience lpdf Bulletin 2804 pdf Bio Rad Hercules Calif Iyer V and Struhl K 1996 Absolute mRNA lev els and transcriptional initiation rates in Saccha romyces cerevisiae Proc Nat Acad Sci U S A 93 5208 5212 Kramer M F and Coen D M 1995 Quantification of transcripts from the ICP4 and thymidine ki nase genes in mouse ganglia latently infected with herpes simplex virus J Virol 69 1389 1399 Morrison T B Weis J J and Wittwer C T 1998 Quantification of low copy transcripts by con tinuous SYBR Green I monitoring during am plification BioTechniques 24 954 962 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods Enzymol 155 335 350 Novoradovskaya N Payette T Novoradovsky A Braman J Chin N Pergamenschikov A Fero M and Botstein D 2000 Pooled high quality reference RNA for human microarrays Strategies 13 121 122 http www stratagene com news newsletter aspx iid 6 Strateg
20. The standard curve plotting function is available in most instrument software If it is not graphing software may be used instead Since the input ng values refer to the input amount of total RNA and not to a known amount of target molecules the numbers generated are simply arbitrary and may not be compared with the numbers calculated for a different GOI If comparison of the relative RNA levels between different RNA targets is desired refer to the efficiency corrected ACt method Basic Protocol 2 or absolute quantification Support Protocol 1 The standard curve method should be used instead of the AACt method Alternate Protocol to find fold changes between samples when the amplification efficiencies of the primer sets are not 100 as determined by the template dilution assay Support Protocol 2 If absolute levels of transcript are desired synthetic RNA standards may be prepared through incorporation of radionucleotides see Support Protocol 1 and used in standard curve analysis Materials 20 ng ul experimental cDNA samples concentration based on RNA input for cDNA synthesis see UNIT 15 5 Dilution series of standard cDNAs see recipe or dilution series of gt S RNA standards see Support Protocol 1 No template control sample NTC prepared at the same time as the cDNA samples using molecular biology grade water instead of RNA see Commentary No reverse transcriptase control samples RT prepared at the same time as the cDNA sa
21. andard deviation stdev 4 Remove any outlier wells from the averaged Ct values gt 0 3 stdev Only one point per replicate may be removed CV may not be used due to the logarithmic nature of both the Ct avg and Ct stdev Instead stdev must be used where a stdev of 0 3 correlates with the maximum allowable standard deviation that can distinguish a two fold change with 99 confidence 0 4 stdev may be used for a 95 confidence interval 1 2 3 4 5 6 7 8 9 ho 11f12 13 14 15 16 17 18 19 20 21 22 23 24 Al1 2 3 4 5 gt 6 7 8 gt Ref Big 10 11 12 113 14 15 16 w LIC 17 18 19 20 ne experimental D Goti TE samples F G GOI 2 H l J GOI 3 K L M GOI 4 N O P 384 well optical reaction plate Figure 15 8 2 Typical plate setup for the AACt method The plate arrangement shown represents an experiment in which 20 samples taken from experimental animals will be assayed for one endogenous control gene and four GOls The samples are plated in triplicate for each of the RNAs assayed Abbreviations GOI gene of interest NTC no template control Ref endogenous control gene The Polymerase Chain Reaction 15 8 9 Current Protocols in Molecular Biology Supplement 73 Table 15 8 2 Master Mixes for the AACt Assay Voum
22. ccurate quantification of transcripts by measurement of 35S incorporation This protocol encompasses template construction in vitro transcription DNase treatment monitoring the efficiency of the reactions by agarose gel electrophoresis and determination of yield To determine yield synthesized RNA can be quantitatively precipitated and purified away from unincorporated nucleotides by application to filters followed by trichloroacetic acid washes This protocol describes a batch precipitation washing method For an individual filtration method see UNIT 3 4 The preparation and use of RNA standards in quantitative RT PCR assays have been described by Kramer and Coen 1995 Materials cDNA or DNA fragment containing target sequence Vector containing T7 T3 or SP6 RNA polymerase promoter Appropriate restriction enzyme UNIT 3 1 for linearizing plasmid 25 24 1 v v v phenol chloroform isoamyl alcohol 49 1 v v chloroform isoamyl alcohol 3 M sodium acetate APPENDIX 2 100 ethanol Nuclease free water 0 5 ug ml sheared salmon sperm DNA 1 M Tris Cl pH 7 5 APPENDIX 2 1 M MgCl APPENDIX 2 1 M DTT APPENDIX 2 500 uM 4NTP mix 500 uM each ATP CTP GTP and UTP 600 Ci mmol 10 mCi ml x S CTP or UTP Bovine serum albumin BSA Spermidine for SP6 only T7 T3 or SP6 RNA polymerase UNIT 3 8 1 U ul RNase free DNase I e g Promega RNeasy Mini Kit Qiagen or equivalent 10 trichloroacetic acid TCA see recipe ice
23. cid targets Real time quantitative PCR is differ ent from end point or in gel analysis see UNIT 15 7 in several ways For real time analy sis the increase in fluorescent signal resulting from PCR product synthesis is recorded dur ing the course of the thermocycle This allows the user to specify the point in the assay at which to read the data Measurements are obtained from the geometric phase of the am plification reaction This is the phase during which all of the components required for the PCR e g dNTPs primers polymerase are in excess and therefore the deficit of an essen tial reaction component will not quench the efficiency of product synthesis Following ge ometric amplification the fluorescence curve reaches a plateau i e the saturation point as the reaction components begin to become limited and the kinetics of the reaction become unpredictable At this stage an increase of one thermocycle no longer correlates with a two fold change in product Applied Biosystems 2002a In contrast in end point PCR quan tification is often obtained by in gel densit ometry measurements at the end or the sat uration point of the reaction where the re action may be at a plateau compromising quantification Real time PCR depends both on a set of universal thermocycling and buffer conditions and on primer efficiency testing and correc tion where necessary As a result the accu racy and precision of the resol
24. d by the manufacturer to be 90 to 99 intact NTP The presence of other con taminating radiolabeled material is taken into account when calculating total input S Stor age time and multiple freezing and thawing cycles will contribute to decomposition of the radionucleotide If the integrity of the reagent is in doubt consult the manufacturer assess the fraction of intact NTP by thin layer chro matography or order a fresh lot While RNA can be quantified by opti cal density care must be taken to eliminate unincorporated nucleotides which will also absorb light at 260 nm and take into ac count RNA secondary structure which can re duce absorbance A protocol for quantifying non radioactive synthetic RNAs that accounts for secondary structure by measuring the ab sorbance of hydrolyzed RNA is described in Iyer and Struhl 1996 This alternative may be suitable for researchers who prefer not to han dle radioactive compounds RNA can also be quantified by comparison with mass standards on a gel however this method may be inaccu rate due to the differential binding of ethidium bromide to single and double stranded RNA regions The most important concern is to avoid RNase contamination of equipment and reagents If RNA yield from transcription is low or undetectable RNA degradation is the most likely culprit Gel electrophoresis of the RNA samples can distinguish transcription failure no products in the pre DNase sample v
25. d the set is considered valid Current Protocols in Molecular Biology t 0 1 with R 0 99 the primer set amplifies at The Polymerase Chain Reaction 15 8 19 Supplement 73 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 20 Supplement 73 Efficiency is dependent upon several factors including pipet calibration primer quality and dilution and even variability in the instrument run The slope therefore may not be exactly 3 3 0 1 In this case the slope of the test primer set must match that of a previously validated endogenous reference gene run for the same standard cDNA within 0 1 For example if the test primer gives a slope of 3 6 and the endogenous reference for the standard cDNA used to test the set gives a slope of 3 5 then the test set is valid However if the endogenous reference primer set gives a Slope of 3 3 and the test primer has a slope of 3 6 the test set is invalid 14 Repeat the linear regression analysis for the TaqMan probe primer set to assess PCR efficiency Melting curve analysis cannot be performed for TaqMan based assays since cleavage of the probe releases the reporter that continuously fluoresces REAGENTS AND SOLUTIONS Use molecular biology grade nucleic acid and nuclease free or sterile filtered double deionized water in all recipes and protocol steps For common stock solutions see APPENDIX 2 for suppliers see APPENDIX 4
26. dard dilution series the GOI copy number values may be interpolated This will allow the user to assess the sensitiv ity i e the lowest detectable copy number of the GOI primer set As an example this type of analysis is used in both clinical and food science for the assessment of pathogen load and gene copy number Pfaffl 2004 By contrast relative quantification does not rely on the knowledge of a given transcript copy number in a standard sample Instead the changes in gene expression or the lev els of a specified transcript may be measured and described as an arbitrary unit relative to some control sample A ACt or standard curve method or to the level of some other control transcript in the same sample standard curve or efficiency corrected ACt method For ex ample relative quantification allows for the measurement of the fold change in expression for gene A in a treated sample versus an un treated sample or for the assessment of the level of gene A in a sample relative to some housekeeping gene in the same sample Critical Parameters and Troubleshooting Primer probe design and validation For RNA analysis it is important to differ entiate message from genomic DNA In this re spect the use of amplicons that span an exon junction allows this requirement to be met This is achieved by designing primer sets with the forward and reverse primers sitting in dif ferent exons However when the intervening intron is
27. dom primers have a much higher probability of efficiently amplifying all RNA transcripts due to their indiscriminate nature ABL pers comm This property also enables RNA secondary structure to be overcome since the priming occurs in random places along the length of the transcript mRNA specific priming by oligo dT s at the poly A tail and any internal poly A tract is another method of RT priming This method will allow only polyadenylated RNAs to be converted to cDNA thus limiting amplification of some GOIs or partially degraded samples A mixture of a random oligo with an oligo dT primer may enhance detection of rare messages while still allowing for the detection of transcripts that lack polyadenylation However the use of this procedure may skew the measurement of relative RNA abundance towards intact full length mRNA over incomplete or rapidly de graded messages Users should decide and test these parameters in their particular experimen tal systems The third method of reverse tran scription priming is the gene specific reverse primer The reverse PCR primer is used to specifically target the GOI for conversion to cDNA This is often performed in the same QPCR plate as the PCR by adding RT en zyme to the PCR mix and adding an incuba tion step prior to the first step in the PCR cy cling program known as one step RT PCR While this may enhance detection of a specific RNA target RNA secondary structure may not be o
28. e for SP6 only If the buffer contains spermidine reaction components other than the enzyme should be at room temperature to avoid precipitation of the DNA If multiple reactions are being performed a master mix containing everything but the template should be prepared In vitro transcription kits can be used for convenience but they will increase the cost Mix reaction components well Transfer 1 ul of the reaction to each of two tubes of salmon sperm DNA from step 4 input S samples Set aside on ice Add 25 to 50 U RNA polymerase T7 T3 or SP6 to the reaction mix reaction volume should now be 98 ul Incubate 1 to 2 hr at 37 C Transfer 5 ul of the reaction mixture to a separate tube and store on ice This is the pre DNase sample that will be assessed by gel electrophoresis Add 5 ul 5 U RNase free DNase I or similar enzyme and incubate at 37 C for 30 min Transcription buffers typically contain sufficient magnesium ion concentrations gt 6 mM to support DNase activity Purify the synthetic RNA using the RNeasy Mini Kit or similar product Perform the final elution twice to maximize yield RNA can also be purified using organic extraction and isopropanol precipitation UNIT 4 1 however this removes less of the unincorporated rNTPs and is especially not recom mended for difficult templates where yields are low e g GC rich sequences Elute or resuspend RNA in 93 ul nuclease free water T
29. e fOr coed Master mix no of Components kinal wolunie per SANA pime is per samples 1 NTC 1 concentration well sample in triplicate 1 extra extra SYBR Green assay 2x SYBR Green mix 1x 5 ul 20 ul 1 1 primer mix 1 25 150 nM each 1 2 ul 4 8 ul uM each primer Template cDNA 10 25 ng 1 25 ul 5 ul H20 N A to 10 ul to 40 ul Total volume 10 ul 40 ul Aliquot 35 ul into each tube containing cDNA TaqMan assay 2x TaqMan mix 1x 5 ul 20 ul 1 1 primer mix 1 25 300 nM each 2 4 ul 9 6 ul uM each primer 5 uM TaqMan probe 250 nM 0 5 ul 2 0 ul Template cDNA 10 25 ng 1 25 ul 5 ul H20 N A to 10 ul to 40 ul Total volume 10 ul 40 ul Aliquot 35 ul into each tube containing cDNA Recommended amount of template for detection of both high and low levels of GOIs If necessary significantly less template may be used picograms Note that the cDNA template quantity is based upon the amount of total RNA input into the reverse transcription reaction 5 6 7 8 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 10 Supplement 73 For each sample normalize the GOI Ct values to those of the reference gene for the same sample according to the equation ACt avgCtgor avgCt ef Calculate the standard deviation of ACt stdev ace as stdevac 4 stdev of reference stdev of GOI Choose a calibrator This will be the sample tissue gene or control group to which the others wil
30. ect sequencing The assay does not tolerate base mismatches be tween primer and template especially in the probe sequence a feature that allows for the detection of single nucleotide polymorphisms SNPs Several primer probe design software packages are available either for purchase or online Otherwise the user may design the primer sets by directly examining the sequence and choosing primers with the correct characteristics as outlined in this protocol The probe is labeled at the 5 end with a fluorescent reporter such as 6 FAM or VIC and at the 3 end with a fluorescent or nonfluorescent quencher The user should consult with the vendor that will synthesize the probe for the availability of each type of label The assay consists of a standard cDNA dilution series from which linear regression curves may be plotted The slope of the resulting curve gives a measure of PCR effi ciency where 3 3 0 1 with a coefficient of determination R of 0 99 indicates a reaction efficiency of 100 Part of the initial SYBR Green validation also includes a melting or dissociation curve analysis At the end of the repetitive cycles of the PCR an additional melt anneal melt cycle is performed The final melt occurs very slowly and the changes in both temperature and fluorescent signal are monitored over time This decrease in fluorescence correlates with the dissociation of the double stranded PCR product releasing the bound SYBR Green I fluorophores
31. een only Add these steps following the 40 cycles of the thermal cycling program 15sec 95 C 15sec 60 C collect data Increase from 60 to 95 C at a 2 temperature ramping rate collect data 15sec 95 C collect data See Support Protocol 2 for a description of the use of melting curve analyses Analyze data 10 Analyze and export raw data see instrument manual for detailed instructions about document setup baseline and threshold settings Some instrument software applications contain a standard curve plotting feature If this function is not available use Excel or another graphing program to plot Ct versus the log nanograms ng of input total RNA for each standard and apply a best fit line to generate the linear regression formula y mx b where y is Ct of the unknown sample m is the slope x is the quantity of the unknown sample in log ng and b is the y intercept for both the reference gene and each GOI Interpolate the unknown sample quantities using the resulting formulas The transformation of the fluorescence signal into Ct data as well as methods for baseline and threshold settings vary by instrument The specific instrument manual should be consulted In analyzing the raw data it is important to adjust the cycle threshold Ct of the am plification plot to within the geometric phase of amplification This is critical for proper analysis because the geometric phase represents the point of the reaction at w
32. een validated The same format is used when testing new probe sets If different standard cDNAs are used to test primers probes on the same plate i e standards derived from different tissues the validated endogenous reference primers probes must also be run for that standard High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 18 Supplement 73 Current Protocols in Molecular Biology Table 15 8 3 Master Mixes for Primer Probe Validation Assays Volume for each Final Volume per cDNA primer mix per Master mix for 8 Components LAS Serd b concentration well sample in triplicate 1 standards 1 extra extra SYBR Green assay 2x SYBR Green mix 1x 5 ul 20 ul 180 ul 1 1 primer mix 1 25 150 nM each 1 2 ul 4 8 ul 43 2 ul uM each primer Template cDNA 0 016 50 ng 1 25 ul 5 ul H20 N A to 10 ul to 40 ul 91 8 ul Total volume 10 ul 40 ul Aliquot 35 ul into each tube containing cDNA TaqMan assay 2x TaqMan mix 1x 5 ul 20 ul 180 ul 1 1 primer mix 1 25 300 nM each 2 4 ul 9 6 ul 86 4 ul uM each primer 5 uM TaqMan probe 250 nM 0 5 ul 2 0 ul 18 pl Template cDNA 0 016 50 ng 1 25 ul 5 ul H20 N A to 10 ul to 40 ul 30 6 pl Total volume 10 pl 40 ul Aliquot 35 ul into each tube containing cDNA Recommended template dilution series The user may modify the range of cDNA concentrations based on the experimental system Note that the cDNA template quantity is based upon the amount of
33. el or equivalent The data analyses are dependent on the type of assay performed and are outlined in detail as part of each protocol NOTE General precautions for working with RNA are described in unr 4 1 and other Chapter 4 units and general precautions necessary for PCR are described in UNIT 15 1 In particular the use of molecular biology grade water RNase DNase nucleic acid free tubes aerosol barrier pipet tips and dedicated pipettors of all types i e pipettors used only for RNA or PCR applications which are kept out of areas used for plasmid or genomic DNA work is strongly recommended for all steps in this unit If dedicated pipettors are not available the available pipettors should be thoroughly cleaned to remove nucleases and potential contaminants such as plasmid or genomic DNA In addition the use of gloves is required since even a small amount of any contaminant can greatly impact the results of the assay STRATEGIC PLANNING To begin performing a QPCR assay design and validation of the appropriate primers and probes must first be completed refer to Support Protocol 2 Second the appropriate assay is selected based on the goal of the experiment and the desired data output refer to Basic Protocols 1 and 2 and the Alternate Protocol for guidelines used in making this determination The chosen assay is then performed and the data are analyzed STANDARD CURVE METHOD FOR RELATIVE QUANTIFICATION The relative standard c
34. ene La Jolla Calif The Polymerase Chain Reaction 15 8 27 Supplement 73 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 28 Supplement 73 Pfaffl M W 2004 Quantification strategies in real time PCR Jn IUL Biotechnology Series No 5 A Z of Quantitative PCR S A Bustin ed pp 87 120 International University Line La Jolla Calif Roche Applied Science 2002 Selection of house keeping genes Technical Note No LC 15 2002 http www roche applied science coml lightcycler online lc_support pdfs Ic_15 pdf Roche Applied Science Indianapolis Ind Schenborn E T and Mierendorf R C Jr 1985 A novel transcription property of SP6 and T7 RNA polymerases Dependence on template struc ture Nucl Acid Res 13 6223 6236 Schmittgen T D and Zakrajsek B A 2000 Effect of experimental treatment on housekeeping gene expression Validation by real time quantitative RT PCR J Biochem Biophys Methods 46 69 81 Shoemaker J P Garland C W and Steinfeld J I 1974 Experiments in Physical Chemistry pp 34 39 McGraw Hill New York Suzuki T Higgins P J and Crawford D R 2000 Control selection for RNA quantitation BioTechniques 29 332 337 Tyagi S and Kramer F R 1996 Molecular bea cons Probes that fluoresce upon hybridization Nat Biotechnol 14 303 308 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Psepe A and Speleman
35. eries just as in Basic Protocol 1 The slope of the resulting standard curve is used as a measure of PCR efficiency E according to the equation E 10 s 9P Note that different GOIs may produce different E values A slope of 3 3 produces an E value of 2 indicating that a perfect doubling of the template has occurred Calculated E values of less than 2 imply that the template has not been perfectly doubled Template primer and probe quality and quantity sample complexity and pipet calibration and buffer conditions like MgCh salt additives and deoxynucleotide concentrations all contribute to this efficiency Pfaffl 2004 see UNITS 15 1 amp 15 5 for further details on these parameters Given optimal buffer conditions and adequate primer probe and sample qualities slight fluctuations in efficiency may still be observed between primer sets run on the same plate or between assay plates even if they have been assembled and run on the same day by the same user Efficiency correction is a means to account for inter or intra assay variability that is attributable to the aforementioned parameters The materials and setup of the assay are the same as in the relative standard curve method see Basic Protocol 1 except that the linear regression formula produced by plotting the Ct versus the log ng of input standard total RNA is used only to determine PCR efficiency This computed efficiency is then used to calculate the RNA levels in arbi
36. ersus loss of RNA during subsequent manip ulations products in the pre DNase sample but not in the purified sample More frequent samples can be taken for gel analysis to help The Polymerase Chain Reaction 15 8 25 Supplement 73 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 26 Supplement 73 pinpoint the troublesome step or reagents A ribonuclease inhibitor such as SUPERase In Ambion may be included in the transcrip tion reaction and added to the purified RNA to preserve RNA integrity however this will merely mask RNase contamination not elim inate it Other possible solutions for poor in corporation or yield could be old reagents Pay particular attention to the freshness of the 35S NTP DTT rNTPs and polymerase A single RNA product species from the transcription is critical for the production of accurate RNA standards Multiple product species of indeterminate content can cause in accuracies in quantification and can also lead to extraneous PCR products Product RNA species longer than expected indicate incom plete cleavage of the parent plasmid Either in creasing the efficiency of the plasmid digestion or purifying the linearized plasmid from an agarose gel will prevent this problem Species smaller than expected could indicate that the RNA polymerases had difficulty transcribing full length RNA Use of a truncated template which eliminates problematic sequences
37. he average stdev and the coefficient of variation CV where CV stdev avg Current Protocols in Molecular Biology 6 Remove outliers normalize the GOI calculate the SD and plot the results see Basic Protocol 1 steps 13 to 17 COMPARATIVE OR AACt METHOD ALTERNATE The comparative Ct or A ACt method is used for measuring the fold changes in expression PROTOCOL of a particular RNA transcript between experimental samples Typically this assay is used when investigating gene expression differences between wild type and knockout or transgenic animals or between vehicle control and drug treated samples The results are then expressed as fold changes relative to a calibrator such as an untreated or wild type sample The AACt method is only applicable when the primer sets for both the GOI and the endogenous reference gene have been shown to give perfect standard curve slopes slopes 3 3 0 1 with R 0 99 as assayed in Support Protocol 2 1 Set up assay as described in Basic Protocol 1 steps 1 to 9a except refer to Figure 15 8 2 for an example of a typical plate setup and to Table 15 8 2 for master mix components Standard cDNA samples are not needed in this method 2 Analyze and export raw data see Basic Protocol 1 step 10 and then import into Microsoft Excel or equivalent spreadsheet program 3 For each of the three replicates of a sample calculate the average avg cycle time Ct and then calculate the st
38. he conditions of the master mixes constant allows not only universal mix conditions but universal cycling conditions If the initial primer set does not perform well new primers are designed also see Support Protocol 2 3 Place the appropriate number of 8 tube PCR strips into a 96 well PCR tube rack see Fig 15 8 1A For convenience use a different color tube strip for each different primer master mix that has been prepared Label the side of each strip with the letter of the row of the reaction plate into which the samples will be placed Alternatively CDNA primer mixes may be made in 0 65 ml microcentrifuge tubes or 0 2 or 0 5 ml PCR tubes The cDNA may be put directly into the optical reaction plates followed by the primer master mix However this is not recommended because it introduces a potential source of experimental error since the assay replicates are not premixed but pipetted individually The end result may be lower assay precision 4 Using a multichannel pipettor put 5 ul of cDNA experimental samples standards and controls into the bottom of the appropriate tubes in the strips Current Protocols in Molecular Biology The Polymerase Chain Reaction 15 8 5 Supplement 73 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 6 Supplement 73 5 Being careful not to touch the cDNA inside the tubes use a multichannel pipettor to place a 35 ul aliquot of the appropriate primer
39. hich Ct is quantitatively related to the amount of initial PCR template Note that a Ct decrease of 1 unit represents a two fold increase in initial PCR template Current Protocols in Molecular Biology It is also important that the coefficient of determination or R value for the linear regression formula be 0 99 If the R value is less than 0 99 this suggests that one or more points of the standard curve are deviating significantly from the best fit line In this case the accuracy of the data obtained from the linear regression formula of this standard curve may be compromised The R value will never be above 0 99 11 Import data into Microsoft Excel or equivalent spreadsheet program with statistical features 12 For each of the three replicates of a sample calculate the average quantity avg of target cDNA interpolated from the standard curve the standard deviation of the average stdev and the coefficient of variation CV according to the formula CV stdev avg 13 Remove any outlier points gt 17 CV After removing the outlier point recalculate avg stdev and CV Only one point per replicate may be removed A 17 CV correlates with the maximum allowable standard deviation that can distinguish a two fold change with 99 confidence when samples are assayed in triplicate wells for both the endogenous reference and the GOI If a 95 confidence interval is acceptable a 21 8 CV may be used as the threshold for removing ou
40. his volume is equal to the volume after step 8 thus making the gel samples from steps and 13 directly comparable Transfer 1 ul of the purified RNA to each of two tubes of salmon sperm DNA from step 4 incorporated S samples Set aside on ice Transfer 5 ul of the purified RNA to a separate tube and store on ice This is the final RNA sample that will be assessed by gel electrophoresis Store the remainder of the RNA at 70 C in multiple aliquots to avoid repeated freeze thaw cycles Current Protocols in Molecular Biology The Polymerase Chain Reaction 15 8 13 Supplement 73 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 14 Supplement 73 The size of the aliquots will depend on their intended use It is convenient to prepare each aliquot with enough RNA for two or three samples Use low retention nonstick microcentrifuge tubes if possible because these prevent the adherence of RNA to the tube walls over time Confirm in vitro transcription product by gel electrophoresis 15 16 17 Electrophorese the pre DNase step 8 and final step 13 samples through a formalde hyde or glyoxal agarose gel see UNIT4 9 of an appropriate concentration for the length of the RNA Visualize synthesized products by staining with ethidium bromide UNIT 4 9 Make the gel as thin as possible to hasten drying later Verify that any plasmid DNA present in the pre DNase sample usually ob
41. ity Order a small scale synthesis of the primers from a suitable vendor Standard de salting of the primers is sufficient and no additional purification e g HPLC is required Once a primer set has been validated large scale synthesis may be more cost effective especially for frequently used primers like the reference genes Validate the primer set according to the remaining steps of this protocol If performing TaqMan based assays validate the primer set before ordering the probe Once the primer set is validated order the dual labeled probe from an appropriate vendor and validate according to the following steps controls and cDNA template series 112 3 4 5 61 71 8 9 10 11 12 13 14 15 16 17 18 19 20121 22 23 24 lt Ref RT NTC 0 06 0 08 gt 0 4 2 10 50 test 1 test 2 test 3 test 4 test 5 test 6 test 7 test 8 test 9 test 10 test 11 test 12 test 13 test 14 test 15 DO ZECAS L O TW MoO wy i being tested primer probe mixes 384 well optical reaction plate Figure 15 8 3 Typical plate setup for primer and probe validation assays The plate arrangement shows a standard cDNA template dilution series that is being used to test 15 primer sets along with an endogenous control primer set that has already b
42. l be compared Find the AACt or calibrated value for each sample according to the equation AACt ACtsample ACtealibrator The stdevy ac will be the same as stdev acs since the calibrator is arbitrarily set to be a constant Find the fold change for each sample relative to the calibrator according to the equation fold change Ca For the sample that is chosen as the calibrator the AACt 0 and therefore the fold change 2 44C Current Protocols in Molecular Biology 9 Plot the resulting fold changes on a bar graph of fold change versus sample name or experimental treatment group Determine the measure of experimental error as SDipid change aa In2 stdev AACt X pe GENERATION OF RNA STANDARDS FOR ABSOLUTE QUANTIFICATION BY REVERSE TRANSCRIPTION PCR Absolute quantification of RNA molecules in unknown samples by reverse transcription PCR RT PCR requires knowledge of the copy number of specific RNA molecules These can be subjected to reverse transcription and PCR in the same manner as the experimental samples thus accounting for the reaction efficiencies of both procedures This protocol describes the production and quantification of synthetic RNA standards for use in Basic Protocol to determine absolute amounts instead of relative levels of a GOI RNA standards are produced via in vitro transcription in the presence of trace amounts of an S labeled ribonucleoside triphosphate rNTP which permits a
43. ld be addressed Transcripts decay at different rates and have variable stability so partial degradation of a sample at any point could lead to complete absence of detection of The Polymerase Chain Reaction 15 8 23 Supplement 73 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 24 Supplement 73 the desired target RNA in a subsequent QPCR assay with little to no change in the house keeping gene used as a control Total RNA is used for QPCR to reduce the number of steps and potential sources of degradation during sample preparation Puri fied messenger or poly A t RNA can also be used However this subtractive purification could lead to the loss of transcripts that do not have a poly A tail or to the preferen tial enrichment of RNAs that have internal A tracts The added processing reduces the re covery of material for subsequent use and can cause degradation If poly A RNA is used 50 to 100 fold less 100 pg sample tem plate is required in the reaction mixture be cause mRNA represents 3 of total cellular RNA Alberts et al 1994 Reverse transcription primers Reverse transcription RT of RNA into complementary DNA cDNA refer to UNIT 15 5 may be performed using several different types of oligonucleotide primers For QPCR the preferred primer is arandom hexamer non amer or dodecamer oligo with 6 9 or 12 base stretches of random sequences respec tively Ran
44. les are plated in triplicate for each of the RNAs assayed and a standard curve is required for each of the RNAs to be measured the row before the unknown samples contains the standard samples whose amounts are shown in ng total RNA Abbreviations GOI gene of interest NTC no template control Ref endogenous control gene RT no reverse transcriptase control the NTC and the RT control as part of the sample group See Figure 15 8 1 for an example of a typical plate setup All steps may be performed at room temperature if a vendor supplied SYBR Green or TaqMan mix is used Most vendor supplied 2 x mixes are stable at room temperature for a number of hours If the instrument is equipped with a plate loading robotic arm several plates may be prepared at once and stacked into the robotic arm queue Alternatively plates may be High Throughput made several days in advance and stored at 4 C until ready to run without a decline in Real Time the performance of the PCR enzyme Advance preparation of plates is not recommended Quantitative if SYBR Green or TaqMan mixes are prepared in the laboratory since the home made ___ Reverse mixes lack additives present in the commercial mixes that confer stability to the reaction P Transcription PCR components 15 8 4 Supplement 73 Current Protocols in Molecular Biology Table 15 8 1 Master Mixes for the Standard Curve and Efficiency Corrected ACt Assays Volume for each cDNA primer mi
45. logy 22 23 24 25 26 27 28 29 30 31 32 33 34 Swirl the beaker on ice for 10 min then pour off the TCA Continual swirling ensures that filters do not clump together CAUTION The TCA and the methanol wash will contain unincorporated rNTPs and should be disposed of as hazardous radioactive waste Repeat steps 21 and 22 twice more Add 50 ml cold methanol to the filters swirl on ice for 5 min and pour off methanol Use forceps to spread out the washed filters on a sheet of plastic wrap and allow to dry A heat lamp can be used to hasten this process Use forceps to transfer these filters to individual scintillation vials Add 5 ml scintillation cocktail to the vials of washed and unwashed filters and measure counts per minute cpm in a liquid scintillation counter The washed and unwashed control filters and the washed input S filters should contain only background levels of radioactivity lt 200 cpm confirming that unincorporated nucleotides were efficiently removed Average the cpm from duplicate filters and subtract background counts input cpm unwashed input S filters unwashed control filters incorporated cpm washed incorporated S filters washed control filters Determine the fraction of S CTP that was incorporated according to the fol lowing equation using volumes at time of sampling ncoeenok incorporated cpm x final RNA volume at step 12
46. luorescent signal If amplification of the NTC occurs primer dimer or other nonspecific PCR products may have been formed or contamination of a reagent or degradation of the primer mix may have taken place If amplification occurs in the RT control this indicates the presence of genomic DNA if the primer probe set does not span an exon junction or the presence of primer dimer or nonspecific PCR products a contaminant or degradation of the primer mix Although all amplification products includ ing those that are nonspecific contribute to the fluorescence signal this fluorescence con tribution can be considered negligible if the Ct values of the NTC and RT are gt 7 cycles dif ferent from the experimental samples Applied Biosystems pers comm If the unknowns are RNA samples the stan dard of choice is an RNA that is reverse tran scribed in the same manner as the unknowns A suitable standard RNA is one in which the expression of the GOI is at a moderate to high level and that has a similar composi tion to the unknown samples The use of a plasmid or linear DNA standard is not ad vised for measurement of an endogenous tis sue transcript because these types of nucleic acids have different background compositions than RNA are extracted differently and are not reverse transcribed and therefore may not have the same RT or PCR amplification efficiency as the experimental samples Ap plied Biosystems 2003 Pfaffl 2004
47. mall pi lot experiment to determine which endogenous reference is appropriate for the particular stud ies A good method for doing this is to perform a AACt assay using several potential normal izer RNAs and a GOI that is not expected to show any fold changes within a small set of samples The GOI is then normalized to each of the reference RNAs individually for all of the samples Finally the housekeeping gene for which there is no detectable fold change of the GOI between the test samples is cho sen for use in experimental assays Applied Biosystems 2001b Guo et al 2001 Roche Applied Science 2002 Controls and relative RNA standards Controls for the assay that are made along side the cDNA standards and unknowns in clude a no template control NTC made by substituting water for RNA and a no reverse transcriptase RT control made by omitting Current Protocols in Molecular Biology the reverse transcriptase For primers that span an exon junction a RT control is not needed for every sample if during the validation pro cess this control shows no amplification prod uct If the system under study is the result of introduction of an expression construct into cells or if the GOI does not have introns or has a known processed pseudogene then RT controls should be made and assayed for ev ery sample An NTC should be run for every primer set of an assay to facilitate the detection of contaminants that contribute to f
48. master mix into each tube 6 Cover the entire rack of tube strips with Parafilm and gently vortex to mix Gently tap or briefly centrifuge the PCR tube racks 2 to 3 min at 1700 x g 4 C or room temperature in a swinging bucket rotor with microtiter plate carriers to get contents to the bottoms of the tubes 7 Using a multichannel pipettor dispense 10 ul of each cDNA primer mix into the appropriate three wells of the optical reaction plate to generate each sample in triplicate as planned in step 1 see Fig 15 8 1B If the multichannel pipettor has 8 or 12 channel dispensing capability the triplicates can be dispensed at the same time for different rows Note that due to the spacing between rows of a 384 well reaction plate every other row can be added at once i e A C E G I K M O and then B D F H J L N P thus greatly minimizing the pipetting time 8 Cover the plate with the optical adhesive cover and then briefly centrifuge the plate as above to get contents to the bottoms of the wells Perform real time PCR 9a For real time PCR Transfer the plate to the real time thermal cycler and run real time PCR using the following program consult the instrument manual for specific instructions 1 cycle 10 min 95 C activates the hot start Tag DNA polymerase 40 cycles 15 sec 95 C collect data throughout 1 min 60 C collect data throughout 9b For melting dissociation curve analysis for use with SYBR Gr
49. may be required Very minor contaminants may not affect quantification by more than a few per cent The researcher can try to estimate the amount of contaminating bands from the gel and account for these in the calculations How ever since the sequence of such contaminants is unknown it is difficult to assess the effect during PCR amplification Thus the authors recommend striving to attain a single product species Additional considerations for constructing the transcription plasmid that will be used to provide the template for run off transcription are as follows 1 The cloned sequence from the gene of interest must include the sequence primed for reverse transcription and the PCR target se quence Ensure that the clone does not include sequences such as introns that are not part of the RNA species Full length cDNA clones yield the most authentic synthetic RNAs In some cases truncated cDNAs may permit more efficient and consistent transcription of a single length RNA product however obtain ing a single length RNA product species is critical 2 If oligo dT is used for reverse tran scription then the synthetic RNA will need a poly A tail In this case construct a tran scription plasmid using a vector such as pSP64 Poly A Promega which contains a run of dA dT residues at the 3 end of the multiple cloning site allowing for the transcription of a synthetic poly A tail 3 Ensure that the orientation of the
50. method see Basic Protocol 2 should be applied Alternatively absolute standards can be generated Support Protocol 1 The efficiency corrected ACt method builds upon the relative standard curve method by incorporating PCR efficiency E into the quantity calculations The standard curve slopes are used to calculate PCR efficiency according to the relationship E 1001109 The efficiency has a maximum value of 2 for perfect doubling of the PCR template see Basic Protocol 2 for an in depth explanation of E Note that in this method the standard curve is used only to determine slope and not to interpolate the RNA values of the unknown samples The efficiency correction is then applied to determine the relative Contributed by Angie L Bookout Carolyn L Cummins Martha F Kramer Jean M Pesola and David J Mangelsdorf Current Protocols in Molecular Biology 2006 15 8 1 15 8 28 Copyright 2006 by John Wiley amp Sons Inc UNIT 15 8 The Polymerase Chain Reaction ES 15 8 1 Supplement 73 BASIC PROTOCOL 1 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 2 Supplement 73 amount of RNA using the measured Ct values for the test samples and is particularly important when comparing the levels of different RNAs whose standard curve slopes deviate from each other by greater than 0 1 This application is useful for comparing the expression profiles of many different RNAs e g those belonging
51. mples using molecular biology grade water instead of reverse transcriptase see Commentary 2x SYBR Green or TaqMan mix containing ROX Applied Biosystems Bio Rad Invitrogen Sigma or see recipe for 2x SYBR Green mix Primer mixes 1 25 uM each forward and reverse primer see recipe and Support Protocol 2 for each reference gene and GOI to be tested 5 uM TaqMan probe for TaqMan protocol only see recipe and Support Protocol 2 Molecular biology grade water nucleic acid and nuclease free 8 tube PCR tube strips optional but recommended can be of low quality since they will only be used for mixing reaction components ISC Bioexpress 96 well PCR tube racks optional but recommended ISC Bioexpress Digital multichannel pipettor 8 or 12 channel 5 to 100 1 capacity recommended Centrifuge with swinging bucket rotor and microtiter plate carriers 384 well optical reaction plates Applied Biosystems Optical adhesive covers Applied Biosystems Real time thermal cycler e g Applied Biosystems 7900HT Microsoft Excel or spreadsheet program with equivalent statistical features Set up plates 1 Plan the plate arrangement according to the number of samples and primer sets to be assayed reference gene plus GOIs For each primer set include the standards Current Protocols in Molecular Biology The Polymerase Chain Reaction 15 8 3 Supplement 73 A sample
52. n Mg concentration will not be appropriate for TaqMan assays This is observed as a decline in PCR efficiency In this case new primers may be designed to flank the probe sequence Additional Materials also see Basic Protocol 1 Primer probe design software Primer Express Applied Biosystems Design primers 1 Retrieve the RNA sequence information from the appropriate source e g Genbank or Ensembl 2 Determine the locations of exon boundaries by aligning the mRNA sequence with its gene or by using NCBI s Entrez Gene Evidence Viewer http www ncbi nlm nih gov or Ensembl s Genome Browser hitp www ensembl org Some genes do not have introns so this step may not be applicable 3 Copy the sequence into the design software Several design programs are available both commercially as stand alone applications and as Web based applications Alternatively primers and probes may be designed by hand Omit this step if designing by hand 4 If using software other than Primer Express use the following parameters a QPCR primers Should have 40 to 60 GC content and melting temperatures around 60 C Should not contain runs of the same nucleotide repetitive sequences or more than two G s and or C s on the 3 end also called GC clamp b PCR product amplicon Should be 50 to 150 bases in length with an approximate melting temperature between 85 and 95 C c TaqMan probe anneals to sequence between
53. number mmm sample number memmmmmmpm 8 OMe OOOE s QO CCS m E 3 DOOMAOOV s ZAO OQQON g z FORO O c aE S D s OE OLLER 5 EIS MBI F S E OKO ADK AI 44 05 Gja OES Bomar TTJJ VVAOAOOM J S O40 42 4344 05 GO K WOE L 96 well PCR tube racks controls and cDNA B template dilution series C hHelsl4 5l6e 7l8 9 elia z0 e 2213 24 A aT _ nto 0 0164 gt 10 gt 50 gt Bl4 2 3 gt gt 7 gt 8 LID 17 gt 18 19 ME experimental aoii LE samples G LIH l J GOI 2 K LIL N GOI 3 O P 384 well optical reaction plate Figure 15 8 1 Typical plate setup for the standard curve and efficiency corrected ACt assays A Organization of PCR tube strips on a 96 well PCR rack when preparing for any QPCR assay It is advisable to premix the cDNA templates with the primer master mixes in tube strips before putting them into the reaction plate This practice decreases the variability between replicate wells A typical cDNA primer mix setup is shown in relation to the final 384 well reaction plate B The plate arrangement shown represents an experiment in which 20 samples taken from experimental animals will be assayed for levels of one endogenous control RNA and three GOls The samp
54. orter and signal is detected Tyagi and Kramer 1996 Whitcombe et al 1999 There are advantages and disadvantages to each type of chemistry SYBR Green versus TaqMan in terms of sensitivity and specificity SYBR Green I binds any double stranded DNA and does not depend on a probe cleavage event Therefore SYBR Green produces ear lier Ct values resulting in an apparent en hanced sensitivity Whittwer et al 1997 Mor rison et al 1998 TaqMan probes on the other hand supply another layer of sequence speci ficity in addition to the forward and reverse primers There are two methods of quantification that may be performed using real time PCR absolute and relative Absolute quantification measures the copy number of a specific nu cleic acid target in a sample Relative quantifi cation measures the difference in copy num ber between two samples that have each been normalized to an endogenous reference Both can be used to compare the effects of different treatments on a particular RNA species or to Current Protocols in Molecular Biology compare the levels of multiple RNA species in a single sample The absolute method requires standards in which the copy number of the particular target has been carefully and accu rately measured From this standard sample a dilution series is made and assayed for the tar get at the same time as the unknown samples From the values obtained from linear regres sion analysis of the stan
55. oscopy requiring to 4 hr depending on number of samples and method of prepara tion Next DNase treat and reverse transcribe RNA to cDNA requiring 3 hr for setup and incubations On the second day prepare mas ter mixes for the assay 30 min to 1 hr Next prepare QPCR plate s 30 min to 1 hr per plate run plate on instrument 1 5 to 2 hr and collect and analyze data 1 to 3 hr For preparation of RNA standards con struction of the transcription plasmid can take anywhere from several days to several weeks one additional day is needed to prepare the template for transcription RNA synthesis gel electrophoresis and scintillation counting can be completed the following day Following overnight gel drying the autoradiography may require several hours to several days for suf ficient exposure During the incubation pe riods for transcription and DNase treatment time becomes available to prepare the gel fil ters and scintillation vials Filters can be spot ted and washed while the gel is running If necessary the samples for gel electrophoresis dissolved in loading buffer and or the sam ples for scintillation counting may be stored at 20 C overnight and analyzed the following day Acknowledgment This work was supported by the Howard Hughes Medical Institute and by grants from the Robert A Welch Foundation I 1275 and the National Institutes of Health U19DK62434 Literature Cited Alberts B Bray
56. pression of the GOI in the experimental samples Current Protocols in Molecular Biology The Polymerase Chain Reaction 15 8 15 Supplement 73 SUPPORT PROTOCOL 2 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 16 Supplement 73 These standard RNAs must be processed in as similar a manner as possible to that of the experimental samples to avoid introducing discrepancies in the efficiency of reverse transcription and PCR amplification of RNA standards versus experimental RNA samples For example mock infected tissue or cell homogenates could be added to RNA standards used for assays of mRNAs from infectious agents Unrelated RNA such as yeast or E coli tRNA may be used at concentrations that mimic the total RNA content of experimental samples in cases where preparations free of the target RNA species are not available If multiple RNA species will be assayed in single experimental samples the different synthetic RNAs may be combined as a single serially diluted RNA standard 35 Employ the resulting duplicate set of standard cDNA samples in the standard curve method see Basic Protocol 1 as the dilution series of standard cDNAs to obtain absolute quantification of RNA species in unknown samples Generate standard curve by plotting Ct against the logarithm of input RNA copy number for the RNA standards Linear regression performed on these points yields an equation from which the copy number of
57. s that are free of contaminating RNases DNases and nucleic acids and follow other guidelines for handling RNA UNIT 4 1 Construct and prepare template for in vitro transcription 1 Construct a transcription plasmid by subcloning UNIT 3 16 placing the sequence contained in the RNA of interest downstream of the promoter for one of the three bacteriophage RNA polymerases T7 T3 or SP6 Isolate the plasmid using a miniprep UNIT 1 6 which will provide sufficient amounts of plasmid DNA for this protocol There are numerous commercially available vectors that contain phage polymerase pro moters on one or both sides of a multiple cloning site see Table 2 10 1 Additional considerations for the construction of this plasmid are discussed in Critical Parameters and Troubleshooting Linearize 10 ug of plasmid with a restriction enzyme UNIT 3 1 that will generate a template for run off transcription Run 5 of the linearization reaction volume on an agarose gel UNIT 2 5A alongside a control sample of uncut plasmid to confirm that the digestion is complete and yields the expected product size s Long transcription products from incompletely digested plasmids can lead to inaccuracies in quantification and extraneous products from RT PCR Purify the linearized template from the reaction mixture using two or three organic extractions with 25 24 1 v v v phenol chloroform isoamyl alcohol UNIT 2 14 one extraction with 49 1 v v
58. served as a low mobility band is absent from the final sample The successful removal of DNA should also be confirmed at the RT PCR stage with a control lacking reverse transcriptase To dry the gel first trim the gel to contain only relevant lanes Stack one sheet of Whatman DE81 paper on top of five sheets of filter paper all cut several inches larger than the gel Center the gel on top of the DE81 paper and cover stack with a sheet of plastic wrap Dry as for a polyacrylamide DNA gel APPENDIX 3A increasing the drying time as needed for the thickness of the agarose gel The DE81 paper should retain most of the unincorporated 5S rNTP while the blotting paper will absorb excess moisture and radioactivity CAUTION Radioactive contamination of the gel dryer may occur Be sure to conduct required surveys and decontamination as directed by the institutional radiation safety committee also see APPENDIX 3F Visualize labeled transcripts by autoradiography or by exposing to a phosphor screen APPENDIX 3A Confirm that there is only one major radiolabeled product of the expected size see Commentary Also confirm that the amount of synthetic RNA represented by the pre DNase sample was efficiently recovered in the final sample If the gel is not run too far it will be possible to visualize the amount of unincorporated a S rNTP removed by the purification procedure Quantify S incorporation and RNA yield 18 19 20 2
59. that the final concentration of the standard is 40 ng ul based on RNA quantity see UNIT 15 5 Include both a no template control NTC for which water is used instead of RNA and a no reverse transcriptase control RT Following the reverse transcription make a 5 fold dilution series of the 40 ng ul standard to obtain working concentration standards of 40 8 1 6 0 32 0 064 and 0 0128 ng ul Store up to 1 month at 4 C or gt 2 years at 20 C with minimal freeze thaw cycles By using 1 25 l well of each of the standards in the final reaction plate the resulting amount of starting template will be 50 10 2 0 4 0 08 and 0 016 ng See Commentary for more information and suggestions about control and standard RNA samples Alternatively if absolute RNA standards are being prepared via Support Protocol 1 these should be serially diluted first and then in parallel with experimental RNA samples DNase treated and reverse transcribed see Support Protocol 1 steps 34 to 35 Current Protocols in Molecular Biology Table 15 8 4 Preparation of 2x SYBR Green Mix Component Volume ul 25 mM MgCl 48 288 600 720 1200 10x Gold PCR buffer 40 240 500 600 1000 10x dNTP mix 40 240 500 600 1000 DMSO 40 240 500 600 1000 1 1000 SYBR Green I 20 120 250 300 500 50x ROX 8 48 100 120 200 AmpliTaq Gold polymerase 5 U l 2 12 25 30 50 H20 2 12 25 30 50 Total volume in 2x buffer 200 1200 2500 3000 5000 SYBR Green mix 2x Combine
60. this case resuspend to 100 uM in Tris Cl pH 8 0 APPENDIX 2 prepared with molecular biology grade water and reagents Before use dilute a small amount of 100 uM stock to 5 uM with more Tris Cl pH 8 0 Store either concentration at 20 C Avoid repetitive freeze thaw cycles and thaw on ice prior to use to preserve the integrity of the probe Protect the probe from excessive exposure to light e g using amber colored screw cap tubes to prevent photobleaching of the fluorescent dyes and evaporation See Support Protocol 2 and Commentary for additional considerations regarding the TaqMan probe The Polymerase Chain Reaction 15 8 21 Current Protocols in Molecular Biology Supplement 73 High Throughput Real Time Quantitative Reverse Transcription PCR 15 8 22 Supplement 73 Trichloroacetic acid TCA solution 10 w v Prepare a 100 w v TCA stock solution by dissolving the entire contents of a newly opened TCA bottle in water e g dissolve the contents of a 500 g bottle of TCA in sufficient water to yield a final volume of 500 ml Store up to 1 year at 4 C Prepare 10 w v TCA by dilution and store up to 3 months at 4 C CAUTION TCA is extremely caustic Protect eyes and avoid contact with skin when preparing and handling TCA solutions COMMENTARY Background Information Quantitative PCR is a rapid robust and highly sensitive polymerase chain reaction method used to quantify specific nucleic a
61. tliers On the other hand the Q test a test for rejection of discordant data may be used to determine outlier points Refer to Shoemaker et al 1974 for a more in depth description of this test 14 For each sample normalize the GOI quantity to that of the reference gene for the sample according to the following equation Use the recalculated values if outlier points were removed in step 13 normalized value se OO avg reference quantity 15 Calculate the standard deviation SD of the normalized value according to the equation SD normalized value xa CV of reference CV of GOL 16 Plot the resulting values as a bar graph of normalized value versus sample name or experimental treatment group with the error bars equal to the SD 17 If desired calculate fold changes between samples by choosing a calibrator sample usually vehicle treated or wild type control and dividing all of the normalized values from step 14 and the SD calculated in step 15 by the normalized value of this calibrator The resulting values are then expressed as fold changes relative to the calibrator sample which should now be equal to 1 EFFICIENCY CORRECTED ACt METHOD The efficiency corrected ACt method is used for determining the relative amounts of dif ferent GOIs that are normalized to an endogenous reference RNA e g 18S cyclophilin It may also be used to determine fold changes of a specific RNA between samples but may be excessive
62. to gene families or related biological pathways The sample space and reagent use limitations mentioned for the standard curve method also apply here The AACt method is the method of choice when the desired output is fold change because standards are not necessary thus saving both reagents and space on the re action plate However this method requires that the amplification efficiencies of the primer probe sets be 100 If the amplification efficiencies are suboptimal but the primers generate a single product as determined by melting curve analysis Support Protocol 2 Basic Protocol 1 should be used to determine fold changes Chapter 4 describes the isolation of RNA from several sources and UNIT 15 5 details the traditional procedure for reverse transcription of RNA into complementary DNA cDNA For QPCR it is recommended that total RNA be resupended in diethylpyrocar bonate DEPC treated water or an equivalent nuclease free buffer that does not contain EDTA The RNA should be treated with DNase and then reverse transcribed using 0 08 ug l final concentration random hexamer or nonamer primers Purified messenger RNA i e poly A RNA unir 4 5 or RNA that has been reverse transcribed using oligo dT or gene specific reverse primers may also be successfully used in the assay See Commentary for more detailed information Following the assay the resulting raw data are analyzed using second party software usually Microsoft Exc
63. total RNA input into the reverse transcription reaction gt For this assay the NTC and RT controls are included as part of the standard sample set Validate primer set 10 Set up assay as described in Basic Protocol 1 steps 1 to 9 except refer to Figure 15 8 3 for an example of a typical plate setup and to Table 15 8 3 for master mix components 11 Test the new primer set using SYBR Green chemistry If valid test the TaqMan based chemistry 12 Following the instrument run first check the dissociation curve If more than one peak is present the primer set is invalid and no other parameters are checked It may be possible to eliminate the nonspecific product s detected in the dissociation curve analysis or gel electrophoresis by addition of enhancers such as betaine UNIT 15 1 This approach may be warranted if the sequence constraints for a given GOI are limiting 13 If a single peak is found in the dissociation curve assess the PCR efficiency by calculating the slope of the linear regression curve as follows a Plot Ct or crossing point CP versus log ng of the standard cDNA input for each concentration of standard as an xy scatter plot This may be performed directly in the instrument software on the Applied Biosystems instrument or can be done in Excel or equivalent b Apply a best fit curve and display the corresponding linear regression formula If the slope of the curve is 3 3 4 100 efficiency an
64. transferable to any system but this must be empirically de termined to ensure the reliability of the data In rare cases the PCR efficiency of a vali dated primer set changes when switched from SYBR Green to TaqMan based chemistry This is due to the difference in primer concen tration and or MgCl concentrations between the two buffers and may often be overcome by redesigning the primers to recognize a se quence around the already synthesized probe Choice of chemistry The selection between SYBR Green I and TaqMan based assays depends upon the RNA sequence If the RNA of interest has no poly morphisms or other variations in the region to which the primers bind and the primers have been correctly designed and validated then SYBR Green based assays are adequate for gene expression analyses It is up to the user to decide if the addition of the often costly TaqMan probe is worth the additional speci ficity it confers In the case of polymorphisms or variants differentiation between different RNA species may require the specificity of the probe which can discriminate a single base difference Template quality The quality of the cDNA template depends upon the integrity of the RNA QPCR will tol erate some degradation of the RNA when ran dom hexamers or other mers are used to prime the reverse transcription reaction How ever it is not good laboratory practice to use degraded RNA and the cause of the degrada tion shou
65. trary units of the GOI and the endogenous control genes The GOI RNA level in each sample is then expressed as a ratio relative to the endogenous control RNA level in that sample Because the data are dependent upon Ct values and not a standard curve the resulting values may be compared to those of another RNA 1 Set up and run assay as described in Basic Protocol 1 steps 1 to 9a Refer to Figure 15 8 1 for an example of a typical plate setup and to Table 15 8 1 for master mix components 2 Analyze and export raw data see Basic Protocol 1 step 10 and then import into Microsoft Excel or equivalent spreadsheet program The threshold values for all RNAs measured including the endogenous reference must be the same It is important to determine a suitable threshold within the geometric phase of the amplification plots for all RNA transcripts to be compared 3 Calculate PCR efficiency E 10 S P for the endogenous control RNA and each GOI from the slopes of their corresponding standard curves 4 Calculate the quantity of the endogenous control RNA and each GOI from their Ct values according to the formula quantity E When the efficiency is 100 i e slope 3 3 and E 2 the equation becomes quantity 2 This serves as the basis for the calculation performed in A ACt method Alternate Protocol 5 For each of the three replicates of a sample calculate the average quantity avg the standard deviation of t
66. urve method is used for determining the level of a gene of interest GOTI relative to an endogenous reference RNA and for calculating relative fold changes of a GOI between experimental samples The assay is useful for determining an expres sion profile of a single GOI within a group of samples A dilution series of standard cDNA samples is constructed for the GOI and reference gene and linear regression analysis is applied The formulas resulting from the standard curves are used to inter polate the GOI and reference gene quantities in the unknown samples An endogenous Current Protocols in Molecular Biology reference gene often a housekeeping gene is used as a control to normalize the amount of input template for each sample see Commentary for parameters used in choosing the appropriate reference gene The data are expressed as normalized RNA level in arbitrary units The type of nucleic acid standard chosen depends upon the nature of the unknown samples and is discussed in the Commentary The relative standard curve assay does not require that the amount of the GOI or reference RNA in the standards be known It depends on the linear regression formula produced by plotting the Ct versus the log nanogram log ng of input standard total RNA It should be noted that cDNA concentrations are not typically determined following reverse transcrip tion of the RNA Here quantity refers to total RNA input prior to reverse transcription
67. ution small est detectable fold change is less than 2 fold whereas resolution for end point in gel measurement is limited to about 10 fold Applied Biosystems 2002a PCR assays in which samples are removed at measured cycle times and electrophoresed and possi bly hybridized have better resolving power than end point analysis However both in gel methods suffer from the lengthy processing steps compared to real time PCR The two most widely used fluorescent de tection methods or chemistries for QPCR are SYBR Green a DNA intercalating dye and the fluorogenic probe Several types of fluoro genic probes are currently available the pop ular dual labeled hydrolysis probe TaqMan probe and the hybridization probes known as molecular beacons and scorpions Both types of probes bind the sequence intervening the forward and reverse primer binding sites and both rely on fluorescence resonance energy transfer FRET to silence the signal from the reporter dye while it is in proximity to the quencher dye A hydrolysis probe is cleaved by the 5 nuclease activity of the polymerase during the primer extension phase of the reac tion and the reporter is released and becomes free to fluoresce continuously Hybridization probes rely on a stem loop structure to keep the reporter and quencher in proximity Upon hybridization to the specific sequence the dis tance between the reporter and quencher be comes too large to silence the rep
68. vercome depending on the priming site and RT efficiency must be considered in addition to PCR efficiency An important assumption that is made when performing RT QPCR is that RT efficiency is similar for the GOI between samples and for different GOIs However different tis sues sample types may contain variable levels of RT inhibiting or enhancing factors Pfaffl 2004 To control for these variables it is rec ommended that all samples to be compared be prepared under the same conditions and at the same time i e with regard to RNA extraction method and RT reaction Endogenous reference gene Normalization of sample loading is essen tial in any quantitative comparative analysis to ensure that the measured differences be tween samples is not attributable to dispro portionate amounts of starting material For gene expression assays the normalizer must be an endogenous gene that is expressed at equal levels in all tissue or cell types and treat ment conditions under study Traditionally one of the so called housekeeping genes e g GAPDH cyclophilin B actin HPRT U36B4 18S rRNA is selected to serve this function The choice is a point of controversy since there are examples of fluctuations for most of the abovementioned genes under various treat ment or physiological conditions Schmittgen and Zakrajsek 2000 Suzuki et al 2000 Guo et al 2001 Vandesompele et al 2002 Dheda et al 2004 The user should run a s
69. will require knowledge of slightly different terminology and pa rameters Livak K J and Schmittgen T D 2001 Analysis of relative gene expression data using real time quantitative PCR and the 2 44 method Meth ods 25 402 408 This article presents a detailed review of the deriva tion of the mathematical applications described in this unit Internet Resources http www ncbi nlm nih gov NCBI Web site http www ensembl org Ensembl Web site http www gene quantification info The Gene Quantification Web site contains a host of information concerning QPCR http pga mgh harvard edu primerbank index html The Primer Bank database hosted by Harvard Uni versity contains user submitted primer sequences for several mouse and human genes http web ncifcrf gov rtp gel primerdb The Quantitative PCR Primer Database QPPD maintained by the National Cancer Institute con tains primer and probe sequences for mouse and hu man genes collected from articles cited in PubMed http www ambion com techlib index html Contains numerous detailed articles and technical bulletins regarding transcription and general RNA handling issues http www promega com techserv Contains technical manuals with detailed protocol tips and troubleshooting for transcription applica tions Other commercial vendor sponsored technical sup port Web sites are also a very good resource for tips about RNA and QPCR applications
70. x per Master mix no of Final Volume per sample in triplicate 1 samples 8 standards Components concentration well extra 1 extra SYBR Green assay 2x SYBR Green mix 1x 5 ul 20 ul 1 1 primer mix 1 25 150 nM each 1 2 wl 4 8 ul uM each primer Template cDNA 10 25 ng 1 25 ul 5 ul H20 N A to 10 ul to 40 ul Total volume 10 ul 40 ul Aliquot 35 ul into each tube containing cDNA TaqMan assay 2x TaqMan mix 1x 5 ul 20 ul 1 1 primer mix 1 25 300 nM each 2 4 ul 9 6 ul uM each primer 5 uM TaqMan probe 250 nM 0 5 ul 2 0 ul Template cDNA 10 25 ng 1 25 ul 5 ul H20 N A to 10 ul to 40 ul Total volume 10 ul 40 ul Aliquot 35 ul into each tube containing cDNA For this assay the no template control NTC and no reverse transcriptase RT control are included as part of the standard sample set gt Recommended amount of template for detection of both high and low levels of GOIs If necessary significantly less template may be used picograms Note that the cDNA template quantity is based upon the amount of total RNA input into the reverse transcription reaction 2 Prepare primer master mixes according to Table 15 8 1 but without template cDNA This can be done in advance and the tubes may be put on ice or stored at 4 C for a few hours before preparing the reaction plate The authors always use the same concentrations of PCR components because this greatly increases the high throughput nature of the assay Keeping t
71. y The results yield data that are highly reproducible and correlate well with tradi tional northern blotting and RNase protection assays Support Protocol 1 will yield 10 to 100 ug of single length transcripts equivalent to 10 to 10 3 RNA molecules with a spe cific activity of 1 x 10 cpm pg Time Considerations In preparation for performing a QPCR as say RNA and subsequent cDNA preparation may be carried out in advance Prior to an actual experimental assay primers and probes must be designed and validated In most cases design and validation may take several days to a few weeks This includes the time to design order and synthesize the primers 2 days depending on the vendor test the primers a few hours and if desired synthesize 7 to Current Protocols in Molecular Biology 14 days depending on the vendor and test a few hours the corresponding probes Once the required primer probe sets are val idated the experimental assays are performed A single plate assay may take 0 5 to 2 hr to pre pare and 2 hr to run The preliminary raw data analyses on the instrument software may take less than half an hour The time required for the following final analyses will depend upon the user s familiarity with both the mathematical and software applications The typical work flow for an experimental assay is as follows On the first day prepare total RNA and de termine concentration by UV or fluorescence spectr

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