GenoExplorer™ miRNA qRT
Contents
1. PART I Extension of miRNA In this step the miRNA is extended Required Materials The following materials are supplied in the GenoExplorer miRNAqRT PCR kit e Poly A extension mix The following materials are supplied by the user 10ng to lug of total RNA Microcentrifuge Heat block or water bath set at 37 C RNase free pipette tips 0 2 and 1 5 ml DNase RNase free microcentrifuge tubes DNase RNase free water Extension Procedure At room temperature add the poly A extension reagents in a 0 2 ml reaction tube 1 Component Amount gt Total RNA 10ng 1ug oo eee X ul e Poly A extension MIX 7771011011 3 ul e DNase RNase free water to 10 ul total X ul total 10 ul 2 Mix gently do not vortex and centrifuge the tube briefly to collect the contents Incubate the tube in 8 heat block or water bath at 37 C for 30 minutes 4 Heat at 95 C for Sminutes cool the reaction on ice and proceed immediately to First Strand cDNA Synthesis U Note Each extended reaction provides enough for five cDNA synthesis reactions PART Il First Strand cDNA synthesis This step is to synthesize the first strand cDNA from the extended miRNAs Required Materials The following materials are supplied in the kit e RT priming mix e Annealing buffer e 2X RT mix The following materials are provided by the user e Thermal cycler or incubator e Microcentrifuge e Ice gt 0 2 and 1 5 ml RNase free microcentrifuge tubes e RNas2. synthesis and SYBR Green I qPCR reagent mix Following isolation of total RNA all the miRNAs in the sample are modified by linking an adaptor at the 5 end using ligase and polyadenlyated using poly A polymerase The universal RT Primer is then used to synthesize cDNA from the tailed miRNA population by reverse transcriptase The first strand cDNA is ready for analysis in qPCR using SYBR Green detection reagents A specific miRNA primer and the universal primer are used for PCR reactions Fig 1 The designed primer sets are optimized to amplify the specific miRNA for either mature or precursor forms Specific primers for any miRNAs in all species can be chosen and ordered from the GenoExplorer miRNA qRT PCR Primer Sets GenoExplorer microRNA qRT PCR Kit Precursor 5 3 EEE 5P Mature mem 3P Mature Expand both ends by ligation and extension Reverse transcribe to cDNA PCR primer design gt gt _ gt 00 gt Fig 1 GenoExplorer microRNA qRT PCR principle and primer design Advantages of the Kit Accurate and Specific Quantification The GenoExplorer miRNA qRT PCR Kit patented provides a simple and highly specific method for miRNA quantification Mature miRNAs situate at either 5 or 3 end or both ends of precursor miRNAs Both ends of miRNA molecules are elongated in this method by 5 ligation and 3 poly A extension This modification allows primer design for specific miRNA
3. Instrument The real time qPCR instrument conditions shown below were executed on an Applied Biosystems ABI 7900 384 well plate system but also apply to an ABI 7500 or an ABI 7300 real time PCR system Other real time instrument may also be applicable Real time qPCR Program Parameters Standard Cycling Program Denature 94 C for 15 minutes 30 45 cycles of Denature 94 C 30 seconds Anneal 59 C 15 seconds Elongate 72 C 30 seconds Elongate 72 C 1 minutes Store 4 C Note annealing temperature may be variable Run the program After cycling hold the reaction at 4 C until further analysis Note on Annealing Temperature This suggested cycling program recommends an annealing temperature of 59 C Increasing the annealing temperature may result in better discrimination of closely related miRNA sequences but with a slight loss in sensitivity 11 GenoExplorer microRNA qRT PCR Kit Appendix Troubleshooting Guide Missing reaction component Repeat reaction setup No an Questionable template quality Analyze starting materials Decrease sample volume Purify DNA Inhibitory substance in reaction either by alcohol precipitation or dialysis Insufficient number of cycles Run additional cycles Incorrect thermocycler programming Verify times and temperatures Errors in block temperature Calibrate heating block Autoclave tubes and use filtered Contaminated tubes or solutions pipette tips Pr
4. isoforms The method generates a miRNA cDNA library The library will be further analyzed for specific mature or precursor miRNAs using PCR Simplified Protocol The system does not need additional chloroform or column purification The simple protocol makes the quantitation achievable within one day All required reagents are included Specific PCR primers however need to be purchased separately based on GenoExplorer microRNA qRT PCR Kit specific miRNA targets refer GenoExplorer miRNA qPCR primer sets Cat 2003 as well as internal reference controls Cat 2004 Flexibility for Real Time or End Point Measurement The assay offers flexibility to do real time or end point PCR measurement by a variety of instrument Product Specification The kit provides enough reagents for 10 cDNA synthesis using total or enriched RNA and 100 PCR reactions cDNA is used for any specific PCR primer pairs Specific primers are available with separate orders Cat 2003 and 2004 GenoExplorer microRNA qRT PCR Kit Kit Components and Storage Conditions e GenoExplorer miRNA qRT PCR Kit Cat 2001 Components Amount Storage Poly A extension mix 30 ul 20 C RT priming mix 80 ul 20 C Annealing buffer 60 ul 20 C 2X RT mix 100 ul 20 C 2X GenoExplorer SYBR Green qPCR mix 750 ul 20 C e GenoExplorer miRNA First Strand cDNA Core Kit Cat 2002 has everything in Cat 2001 but not 2X SYBR Green q
5. well and incubate at 37 C for 30 minutes Heat at 95 C for 5 min and cool on ice Part II First Strand cDNA Synthesis Procedure Add the following reagent in a 0 2 ml DNase RNase free tube on ice e Extended RNA from previous step Part I 2 ul e RT priming mix asssssssssrserrrressese 8ul total 10 ul Incubate at 46 C for 10 min Chill on ice and add the following e 2X first strand mix 07 10 ul total 20 ul Incubate at 42 C for 60 min Heat at 95 C for 5 min Chill on ice or store at 20 C Part III qPCR Procedure Directly use previous cDNA or dilute it 1 10 see detailed protocol e cDNA template from previous step Part II 2 ul e 2X GenoExplorer SYBR qPCR mix 7 5 ul e Forward primer veccaeiscscicidanessnabedsceerontes I ul e Reverse Primer sous piscavaetiwe nw Gii nscevewess 1 ul e DNase RNase free water eee eee 3 5 ul total 15 ul Perform PCR using either real time or end point measurement Standard Cycling Program Denature 94 C for 15 minutes 30 45 cycles of Denature 94 C 30 seconds Anneal 59 C 15 seconds Elongate 72 C 30 seconds Elongate 72 C 1 minute Store 4 C Note Annealing temperature may be variable GenoExplorer microRNA qRT PCR Kit GenoExplorer miRNA qRT PCR Detailed Protocol General Description GenoExplorer miRNA qRT PCR system employs elongation of both ends of miRNAs by a 5 end ligation and 3 poly A extension approach followed by reverse tran
6. 6 GenoSensor Corporation GenoExplorer miRNA qRT PCR Kit for Catalog s 2001 2002 2003 2004 Version A April 2007 GenoExplorer microRNA qRT PCR Kit Table of Contents hirden 2000 eens 2 sake Gees 2 Kit Components and Storage Condition 0 2 011111111111 5 Additional Required 6 Related Products from GenoSensor 020 echo wnnie ceed siaweatiepeus 6 GenoExplorer miRNA qRT PCR Quick Start Protocol ssrrenerorervrnrnvnnnvenvvenrernenenvenveenr 7 GenoExplorer miRNA qRT PCR Detailed Protocol 7 8 0 OEE TEES EEEREN EEG 12 0 gunnaemeeeeatate 12 Technical SEE Jepsen etrkni kaka EE eE EEEE 13 Literature Citation When describing a procedure for publication using these products we would appreciate that you refer to them as the GenoExplorer miRNA qRT PCR Kit Patents and Trademarks GenoExplorer is a trademark of GenoSensor The GenoExplorer miRNA qRT PCR Kit and the GenoExplorer miRNA Primer Sets are covered by patents pending GenoExplorer microRNA qRT PCR Kit Introduction Overview The GenoExplorer miRNA qRT PCR System is a highly robust and efficient system for amplifying specific microRNA molecules or other small non coding RNA from small quantities of total RNA to generate sufficient amounts of material for downstream study This system is engineered for use with as little as 10 ng of isolated total RNA as
7. PCR mix e GenoExplorer miRNA qPCR Primer Sets Cat 2003 Components Amount Storage Forward primer specific miRNA primer 100 ul 20 C Universal reverse primer 100 ul 20 C e GenoExplorer miRNA qPCR Reference Primer Sets Cat 2004 Components Amount Storage Forward primer specific reference primer 100 ul 20 C Universal reverse primer 100 ul 20 C Shipping and Storage GenoExplorer miRNA qRT PCR kits are shipped on dry ice Components should be stored at temperatures shown in the above table At proper storage conditions components are stable for 1 year from the date received Expiration dates are also noted on product labels Safety Warnings and Precautions For research use only Not recommended or intended for the diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Consider all chemicals as potentially hazardous Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products Wear suitable protective clothing such as laboratory overalls safety glasses and gloves Exercise caution to avoid contact with skin or eyes if contact should occur wash immediately with water Material Safety Data Sheet for products is available upon request GenoExplorer microRNA qRT PCR Kit Additional Required Materials Total RNA containing the small RNA RNa
8. e free pipette tips GenoExplorer microRNA qRT PCR Kit First Strand cDNA Synthesis Procedure 1 Centrifuge the 10 ul of extended RNA from previous step and place on ice Use 2 ul of the 10 ul for the following step 2 Component Amount e Extended RNA from previous step Part I 2 ul e RT priming ASer 8 1 3 Incubate the tube at 46 C for 10 minutes 4 Place the tube on ice for I minute Note amp In most cases Annealing Buffer is not required If non specific PCR products are seen adding the Annealing Buffer and reducing the RT priming mix to keep final volume of 10 ul are recommended gt This procedure is for 8 single reaction However for multiple reactions prepare 8 master mix with 8 5 overage for accurate pipetting 5 Add the following to the tube to a final volume of 20 ul 6 2 MIX eee 10 ul Briefly spin the tube briefly to collect the contents 7 Transfer the tube to a thermal cycler preheated to 42 C and incubate for 60 minutes Incubate at 95 C for 5 minutes to stop the reaction then chill the reaction on ice 9 Store aliquots at 20 C or proceed immediately to qPCR oo Note For starting material of 100ng or less of total RNA use 2 ul of cDNA from previous step for qPCR reaction For amounts of lug or more of total RNA make 1 10 dilution of cDNA by adding DNase RNase free water to 200 ul final volumes and use 2ul of the diluted cDNA for qPCR reaction For amount of 100ng lp
9. g total RNA dilution should be based on the abundance of your target RNA in the sample PART III qPCR This step is to quantitatively amplify specific miRNA transcripts from the first strand cDNA The PCR can be performed by either real time or end point Required Materials The following materials are supplied in the kit e 2X GenoExplorer SYBR qPCR mix The following materials are needed to prepare or purchase separately e Specific PCR primers purchase GenoExplorer miRNA qPCR Primer Sets Cat 2003 e Reference control primers purchase GenoExplorer miRNA qPCR Reference Primer Sets Cat 2004 The following materials are provided by the user 10 GenoExplorer microRNA qRT PCR Kit Thermal cycler Microcentrifuge Ice 0 2 and 1 5 ml RNase free microcentrifuge tubes RNase free pipette tips DNase RNase free water PCR Procedure l 2 3 Add the following components to each DNase RNase free PCR tube or plate well Component Amount e cDNA template from step Part II or diluted 2 ul e 2X GenoExplorer SYBR qPCR ee 7 5 ul e Forward primer 3 UM nee eee eee eee e ee eeeeeaeeees I ul gt Reverse Primer FM I ul e DNase RNase free water 3 5 ul total 15 ul Cap or seal the tube plate and gently mix Make sure that all components are at the bottom of the tube plate Centrifuge briefly if needed Place reactions in a preheated real time instrument Suggested Cycling
10. imer Annealing temperature too High Lower annealing temperature in 2 C increments Premature Taq DNA polymerase Add components on ice chilled Non specific replication place PCR reaction mixtures to Products a preheated 94 C thermocycler Primer Annealing temperature too Raise annealing temperature in 2 low increments Insufficient mixing of reaction buffer Reaction buffer must be thoroughly mixed prior to use FASER pues Reduce Forward and Reverse primer concentration to 0 1 0 4 uM Fe Reet ME Reduce RT Priming mix to 8 4ul and bring the volume to 8ul with Water Use non aerosol tips Exogenous DNA contamination Set dedicated area for reaction setup Wear gloves GenoExplorer microRNA qRT PCR Kit Technical Service For more information or technical assistance please call write fax or email GenoSensor Corporation 4665 S Ash Avenue Suite G 18 Tempe Arizona 85282 Tel 1 480 598 5378 Fax 1 480 755 3319 Email tech_service genosensorcorp com Web www genosensorcorp com Limited Warranty GenoSensor is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about a GenoSensor product or service please contact our Technical Service at tech_service genosensorcorp com GenoSensor warrants that all of its products will perform acco
11. rding to the specifications stated on the certificate of analysis This warranty limits GenoSensor Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions GenoSensor reserves the right to select the method s used to analyze a product unless GenoSensor agrees to a specified method in writing prior to acceptance of the order GenoSensor makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore GenoSensor makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service GenoSensor assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 13
12. scription This offers greater selectivity for PCR primer design and specificity to quantify either mature or precursor miRNA forms The amplified PCR is specific for individual forms without mixture of both The modified miRNAs are then reverse transcribed to cDNA Specific miRNAs are amplified and quantified by PCR Handling RNA Samples When working with RNA always use proper microbiological aseptic techniques Use RNase and DNase free reagents water glassware and plasticware Use non powdered gloves during all steps of sample labeling chip hybridization washing detection and scanning RNA Preparation Total RNA or enriched small RNA samples are recommended In this protocol starting from total RNA 10ng lug was described High quality and sufficient amounts of RNA samples is crucial for experiments with microarrays RNA quality can be evaluated by visualizing the RNA on a gel as well as by calculating the A A ratio On a denaturing gel or on an ordinary agarose gel in denaturing buffer the RNA should appear as two bright distinct bands that represent the 285 and 18S ribosomal species The 285 band should be brighter than the 18S band Tailing of these major bands down the gel or a background smear behind these bands that gets heavier at lower molecular weights can indicate degradation of the RNA Degraded RNA will produce high background and low signal intensity microarray results GenoExplorer microRNA qRT PCR Kit
13. se free water Adjustable pipettors RNase free tips RNase free polypropylene microcentrifuge tubes 0 2 0 5 or 1 5 ml Graduated cylinder Microcentrifuge Incubator set at 37 C Incubator set at 42 C Incubator or heating block set at 75 C Heating block at 95 C Thermocyclers real time feature is optional Minicentrifuge Electrophoresis system optional The GenoExplorer qRT PCR kit is open to a variety of equipment The examples given are only suggestions rather than specific recommendations Please contact technical support if you have specific questions Related Products from GenoSensor GenoExplorer microRNA Array Full Kit Cat 1101 1199 GenoExplorer microRNA Array Labeling Kit Cat 1301 GenoExplorer microRNA Biochips Kit Cat 1201 1299 GenoExplorer microRNA Probe Set Cat 1401 1499 GenoExplorer Reagents for Hybridization Assay Cat 1501 1504 GenoExplorer microRNA qRT PCR Kit Cat 2001 GenoExplorer microRNA First Strand cDNA Core Kit Cat 2002 GenoExplorer microRNA qPCR Primer Sets Cat 2003 GenoExplorer microRNA qPCR Reference Primer Sets Cat 2004 GenoExplorer microRNA qRT PCR Kit Quick Start Protocol Part I Extension Procedure Add the following reagent in a 0 2 ml DNase RNase free tube on ice e Total RNA 10ng Ipg eee X ul e Poly A extension eee eee 3 ul DNase RNase free water to 10 ul total X ul total 10 pl Mix
14. starting material The procedure is simple and requires no purification and allows for detection of either mature or precursors microRNAs in the sample in less than three hours MicroRNAs are a class of non coding single stranded RNA molecules that regulate their targets by translational inhibition and mRNA destabilization Increasing evidence suggests that microRNAs may also play a major role in cellular transformation and carcinogenesis by acting either as oncogenes or tumor suppressors They are transcribed as primary miRNAs which are then processed to a shorter hairpin pre miRNAs approximately 70 90 nucleotides structures by a nuclear enzyme Drosha then further processed by a cytoplasmic enzyme Dicer RNAse 111 1186 endonuclease to approximately 20 22 nucleotides single stranded Mature miRNAs Despite hundreds of miRNAs that have been discovered the actual mechanism by which they regulate cellular functions such as regulation of developmental timing pattern formation and secretion are not well understood Several aspects of miRNAs made them elusive and difficult to detect and study such as their small size lack of poly adenylated tails their propensity to bind targets with imperfect sequence homology and also their sequence homology within families the detection technology must be able to distinguish sequences that differ only by 1 2 nucleotides Product System Workflow The GenoExplorer miRNA qRT PCR kit contains the first strand cDNA
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