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Sample & Assay Technologies miScript® miRNA PCR

1. 29509 OL TON TOL JOL JOL L 8888523322223882 00 A O OOO TOL OLIS I LOO 76s56 959 9 9 06 00 1 gt 8182019 10 0 10 200 8302916 9 0 10 0 BSElSRIRF F553 SL ROO 8592829229382 896295s9s9s9992900 059 0 0 sR ROG 4 O 0 EL gt 2 Reverse Positive transcription PCR control snoRNA snRNA C elegans miR 39 miScript miScript PCR Controls Figure 4 Pathway Focused miScript miRNA PCR Array layout for plate formats E G Pathway Focused miScript miRNA PCR Arrays in formats E and G include 4 replicates of the same assays as provided in the 96 well format shown in Figure 3 control Primer Assay miScript miRNA PCR Array Handbook 05 2012 16 02 03 04 05 06 07 08 09 GIO Giz Gis p GIS 16 17 18 19 20 21 22 23 24 25 26 27 29 30 31 32 33 34 35 36 3 38 39 40 4 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 6465166 6768607 72 73 74 75 76 97 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 14
2. AVG CPS correction factor If this value is less than 7 then no inhibition of the reverse transcription reaction is apparent No action is needed If this value is greater than 7 there is evidence of impurities that may have inhibited the reverse transcription reaction See the Troubleshooting Guide page 46 Note As the cDNA will be divided among different numbers of wells in the downstream experiment a correction factor is introduced into the calculation according to the number of plates Rotor Discs that will used This corrects for the dilution of the miRTC The calculation is performed as shown in Table 12 miScript miRNA PCR Array Handbook 05 2012 43 Table 12 PO calculations for miScript miRNA QC PCR Arrays Intended use of Correction A Cj life calculation cDNA factor Pathway Focused 1 5 1 5 AVG miScript miRNA PCR Array miScript miRNA HC AVG 0 5 Whole miRNome 1 x 0 5 AVG 384 well plate or 4 x 96 well plates Rotor Discs Whole miRNome 2 x AVG CTC 1 5 384 well plate or 8 x 96 well plates Rotor Discs Whole miRNome 3 x AVG 2 0 AVG 384 well plate or 12 x 96 well plates Rotor Discs Whole miRNome 4 x 2 5 AVG 384 well plate 16 x 96 well plates Rotor Discs 8 values of the miScript Primer Assa
3. 24 25 26 27 28 29 30 31 32 33 34 35 36 D 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 COB BB BBD lt om C elegans snoRNA snRNA Reverse Positive miR 39 miScript miScript transcription PCR Primer Assay PCR Controls control control Figure 3 Pathway Focused or miRNome miScript miRNA PCR Array layout for plate formats A C D Wells to G12 1 84 each contain a miScript Primer Assay for a pathway disease functionally related mature miRNA Wells and H2 contain replicate C elegans miR 39 miScript Primer Assays that can be used as an alternative normalizer for array data Ce Wells H3 to H8 each contain an assay for a different snoRNA snRNA that can be used as a normalization control for the array data SN1 SNORD61 assay 532 SNORD68 assay SN3 SNORD72 assay SN4 SNORD95 assay SN5 SNORD964A assay SN6 RNU6B RNU6 2 assay Wells H9 and H10 contain replicate miRTC miScript Primer Assays miRTC Wells H11 and H12 contain replicate positive PCR controls PPC miScript miRNA PCR Array Handbook 05 2012 15 OL JOL JOL JOLIS IOL I SEERR SRE EEF ER 3 amp 6 amp 8 G8 360 6 6 96 00 z
4. 1 1 AVG 1 1 AVG 2 AVG Cy _ 2 1 AVG C4 27 AVG _ 2 7 AVG C 3 1 AVG 3 1 AVG For miScript miRNA HC PCR Arrays use the calculation shown for 1 x 384 well plate 8 10 value for each mature miRNA profiled in the plate is calculated using the formula AC C AVG 2 3 4 5 6 Note Choose an appropriate snoRNA snRNA control for normalization Six snoRNA snRNA controls SN1 6 are included on each array Make sure that the selected controls are not influenced by the experimental conditions If one or more snoRNA snRNA have been previously independently identified and if the miScript miRNA PCR Array reproduces those results use the average of their C values in the equation above If an appropriate snoRNA snRNA has not been previously identified use the average C4 value of all the snoRNA snRNA When biological and or technical replicates are performed calculate the average AC value of each snoRNA snRNA each well across those replicate arrays for each treatment group AAC for each miRNA across 2 miScript miRNA PCR Arrays or 2 samples is calculated using the formula AAC AC sample 2 AC sample 1 where sample 1 is the control sample and sample 2 is the experimental sample Fold change for each gene from sample 1 to sample 2 is calculated as 2624m miScript miRNA PCR Array Handbook 05 2012 37 Optional If the fold change is
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6. Tel 49 6131 19240 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of miScript II RT Kit and miScript SYBR Green PCR Kit is tested against predetermined specifications to ensure consistent product quality 8 miScript miRNA PCR Array Handbook 05 2012 Introduction The miScript PCR System consists of the miScript 1 RT Kit miScript PreAMP PCR Kit miScript miRNA PCR Array miScript Primer Assay miScript SYBR Green PCR Kit and miScript miRNA PCR Array data analysis tool The miScript PCR System allows sensitive and specific detection and quantification of microRNA miRNA The miScript PCR System uses total RNA that contains miRNA as the starting material for cDNA synthesis and separate enrichment of small RNA is not needed A single cDNA preparation can be used with a miScript miRNA PCR Array to rapidly profile the expression of mature miRNAs miScript miRNA PCR Arrays are mature miRNA specific forward primers miScript Primer Assays that have been arrayed in biologically relevant pathway focused and whole miRNome panels These PCR arrays are provided in ready to use 384 well plate 96 well plate and 100 well Rotor Disc formats miScript miRNA PCR Arrays which are available for several species provide guaranteed high performance and are fully customizable Each assay in a miScript miRNA PCR Array has been verified to ensure sensitive and specific detection of mature miRNA by real time PCR
7. Wells 6 to 8 each contain an assay for a different snoRNA Well 6 SNORD61 assay Well 7 SNORD95 assay Well 8 SNORD96A assay Wells 9 and 10 contain replicate miRTC miScript Primer Assays miRTC Wells 11 and 12 contain replicate positive PCR controls PPC This pattern is repeated 7 additional times from wells 13 to 96 Wells 97 to 100 are empty This format enables the quality assessment of up to 8 cDNA samples Controls in miScript miRNA PCR Arrays and miScript miRNA QC PCR Arrays The final 12 wells of each miScript miRNA PCR Array contain controls The purpose of the controls in miScript miRNA PCR Arrays and the miScript miRNA QC PCR Array is detailed below and in Table 1 miScript miRNA PCR Array Handbook 05 2012 21 Table 1 Controls in each miScript miRNA PCR Array Control Purpose C elegans miR 39 miScript Primer Alternative data normalization using Assay exogenously spiked Syn cel miR 39 miScript miRNA Mimic 3 mature miRNA miScript Primer Ubiquitously expressed mature miRNAs Assays miR 16 miR 21 miR 191 6 snoRNA snRNA miScript PCR Data normalization using the AAC Controls method of relative quantification SNORD61 assay SNORD95 assay SNORD9Y6A assay SNORD68 assay SNORD72 assay RNU6B RNU6 2 assay miRNA reverse transcription control Assessment of reverse transcription miRTC miScript Primer Assay performance Positive PCR control PPC Assessment of PCR performance Only included on
8. f using a Roche LightCycler 480 adjust the ramp rate to 1 C s Due to software requirements the fluorescence detection step must be at least 30 s with the ABI PRISM 7000 or 34 s with the Applied Biosystems 7300 and 7500 If using a Roche LightCycler 480 use 45 cycles 8 Place the plate Rotor Disc in the real time cycler and start the cycling program 9 Perform data analysis miScript miRNA PCR Array Handbook 05 2012 41 Protocol Data Analysis for Quality Control Using miScript miRNA QC PCR Arrays This protocol describes the steps for analysis of data from miScript miRNA QC PCR Arrays The first steps should be performed by the user The later steps are performed by the free data analysis software A free miScript miRNA QC PCR Array Data Analysis Excel Template is available at http pcrdataanalysis sabiosciences com mirna This tool provides a summary of the data from the miScript miRNA QC PCR Array and interprets the miRNA reverse transcription control and positive PCR control If cDNA was preamplified using the miScript PreAMP PCR Kit and a miScript PreAMP Primer Mix refer to the miScript PreAMP Handbook for a data analysis protocol Important point before starting Text marked with a denotes instructions for 96 well and 384 well plates formats A C D and text marked with a A denotes instructions for 100 well Rotor Discs format R Procedure Steps performed by the user 1 Define the bas
9. or c materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes Commercial Purposes means any activity for which a party receives or is due to receive consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research The buyer cannot use this product or its components or materials made using this product or its components for therapeutic diagnostic or prophylactic purposes Further information on purchasing licenses under the above patents may be obtained by contacting the Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 Email outlicensing lifetech com NOTICE TO PURCHASER LIMITED LICENSE The purchase of this product miScript SYBR Green PCR Kit includes a limited non transferable right to use the purchased amount of the product to perform Applied Biosysterns patented Passive Reference Method for the purchaser s own internal research No right under any other patent claim and no right to perform
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11. 100 miScript miRNA PCR Array add 20 ul per well 40 miScript miRNA PCR Array Handbook 05 2012 5 Carefully tightly seal the miScript miRNA QC PCR Array with Optical Thin Wall 8 Cap Strips Formats A and D Optical Adhesive Film Formats C E F and G or Rotor Disc Heat Sealing Film Format R 6 Centrifuge the PCR plate for 1 min at 1000 g at room temperature 15 25 C to remove bubbles Note This step is not necessary for reactions set up in Rotor Discs 7 Program the real time cycler according to Table 11 Note Perform dissociation curve analysis of the PCR product s to verify their specificity and identity Dissociation curve analysis is an analysis step built into the software of real time cyclers Follow the instructions provided by the supplier Table 11 Cycling conditions for real time PCR Step Time Temperature Additional comments PCR 15 min 95 HotStarTag DNA Polymerase is Initial activated by this heating step activation step 3 step Denaturation 155 Annealing 30 s Extension 30 s Perform fluorescence data collection Cycle number 40 cycles Cycle number depends on the amount of template cDNA and abundance of the target For Bio Rad models CFX96 and CFX384 adjust the ramp rate to 1 C s t For Eppendorf Mastercycler ep realplex models 2 25 4 and 45 for the Silver Thermoblock adjust the ramp rate to 26 for the Aluminum Thermobock adjust the ramp rate to 35
12. Due to the hot start it is not necessary to keep samples on ice during reaction setup or while programming the real time cycler Table 10 Reaction mix for miScript miRNA QC PCR Arrays Array format 384 well 96 well Rotor Disc 100 Component Formats E G Formats C D Format R F 2x QuantiTect 75 ul 175 ul 150 ul SYBR Green PCR Master Mix miScript 15 ul 35 30 ul Universal Primer RNase free water 55 ul 135 ul 115 ul Template cDNA 5 ul 5 ul 5 ul Total volume 150 pl 350 pl 300 yl These volumes provide 10 ul per well reaction volume for a 384 well plate 25 ul per well reaction volume for a 96 well plate and 20 ul per well reaction volume for a 100 well Rotor Disc t Volumes shown are sufficient for one cDNA template In total 8 cDNA samples can analyzed on one 96 well miScript miRNA QC PCR Array 8 cDNA samples can be analyzed on one Rotor Disc 100 miScript miRNA QC PCR Array and 32 cDNA samples can be analyzed on one 384 well miScript miRNA QC PCR Array No optimization of the Mg concentration is required The final concentration provided by 2x QuantiTect SYBR Green PCR Master Mix gives optimal results 3 Carefully remove the miScript miRNA QC PCR Array from its sealed bag 4 Add reaction mix to the wells of the miScript miRNA QC PCR Array as follows For 384 well miScript miRNA PCR Array add 10 ul per well For 96 well miScript miRNA PCR Array add 25 ul per well For Rotor Disc
13. and or is greater than 21 for 96 well and 384 well plates or 17 for 100 well Rotor Discs a Variation in instrument Different instruments have different levels of sensitivity sensitivity If an average lt value of 19 2 for 96 well and 384 well plates or 15 2 for 100 well Rotor Discs is difficult to obtain for the instrument used the observed average value should be acceptable as long as it does not vary by more than 2 cycles between arrays being compared b HotStarTaq DNA Be sure that the initial heat activation step at Polymerase not 95 C took place for 15 minutes and that all activated with a hot other cycle parameters were performed start according to the protocol 46 miScript miRNA PCR Array Handbook 05 2012 Comments and suggestions c Poor quality RNA that Check the A260 A280 and A555 A423o ratios of the may contain PCR RNA samples Be sure to perform the dilutions inhibitors for spectrophotometry in RNase free 10 mM Tris Cl pH 7 5 If necessary repurify RNA with a spin column based clean up method such as the miRNeasy Mini Kit cat no 217004 No product or product detected late in real time PCR indicative of problems occurring during reverse transcription Pipetting error Check the pipets used for experimental setup missing reagent when Mix all reagents well after thawing and repeat setting up reverse the reverse transcription reaction transcription reaction b Incorrect setup o
14. can be used to assess the quality of cDNA samples prior to running a miScript miRNA PCR Array see protocol page 39 Procedure 1 Thaw 2x QuantiTect SYBR Green PCR Master Mix 10x miScript Universal Primer template cDNA and RNase free water at room temperature 15 25 Mix the individual solutions 2 Prepare a reaction mix according to either Table 6 for a Pathway Focused miScript miRNA PCR Array or Table 7 for a miRNome miScript miRNA PCR Array Mix thoroughly but gently Due to the hot start it is not necessary to keep samples on ice during reaction setup or while programming the real time cycler Table 6 Reaction mix for Pathway Focused miScript miRNA PCR Arrays Array format 384 well 4 x 96 well Rotor Disc 100 Component 96 Formats C D Format R Formats E G 2x QuantiTect 550 1375 yl 1100 ul SYBR Green PCR Master Mix 10x miScript 110 275 220 Universal Primer RNase free water 340 ul 1000 ul 780 ul Template cDNA 100 ul 100 ul 100 ul Total volume 1100 pl 2750 yl 2200 yl Volumes shown are sufficient for one cDNA template In total 4 cDNA templates can be analyzed on one 384 well Pathway Focused miScript miRNA PCR Array because assays are arrayed in quadruplicate see Figure 4 page 16 t No optimization of the concentration is required The final Mg concentration provided by 2x QuantiTect SYBR Green PCR Master Mix gives optimal results Provides 0 5 1 ng cDNA
15. commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Limited License Agreement for the miScript PCR System Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties Other than expressly stated licenses QIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties This kit and its componen
16. each contain an assay for a different snoRNA snRNA that can be used as a normalization control for the array data SN1 SNORD61 assay SN2 SNORD68 assay SN3 SNORD72 assay SN4 SNORD95 assay SN5 SNORD96A assay SN6 RNU6B RNU6 2 assay Wells P21 and P22 contain replicate miRTC miScript Primer Assays miRTC Wells P23 and P24 contain replicate positive PCR controls PPC miScript miRNA PCR Array Handbook 05 2012 17 Empty N PPC miRTC 2 SN 2 1 6 Postive PCR control Ce Reverse transcription control Bl snoRNA snRNA miScript PCR Controls C elegans miR 39 miScript Primer Assay o Figure 6 miScript miRNA PCR Array layout for Rotor Disc format R Wells 1 to 84 each contain a miScript Primer Assay for a pathway disease functionally related gene Wells 85 and 86 contain replicate C elegans miR 39 miScript Primer Assays that can be used as an alternative normalizer for array data Ce Wells 87 to 92 each contain an assay for a different snoRNA snRNA that can be used as a normalization control for the array data SN1 SNORD61 assay SN2 SNORD68 assay SN3 SNORD72 assay SN4 SNORD95 assay SN5 SNORD964A assay SN6 RNU6B RNU6 2 assay Wells 93 and 94 contain replicate miRTC miScript Primer Assays miRTC Wells 95 and 96 contain replicate positive PCR controls PPC Wells 97 100 are empty miScript miRNA QC PCR Array plate layout miScript miRNA QC PCR Arrays enable assessment of the qual
17. greater than 1 the result may be reported as a fold upregulation If the fold change is less than 1 the negative inverse of the result may be reported as a fold downregulation 11 Fold changes are presented by the data analysis tool in a variety of formats including a tabular format a scatter plot a three dimensional profile and a volcano plot when replicates are included 38 miScript miRNA PCR Array Handbook 05 2012 Protocol cDNA Quality Control Prior to Profiling Mature miRNA Use of the miScript miRNA QC PCR Array enables testing of the quality of cDNA prepared using miScript HiSpec Buffer saving time and reagents Only 5 ul diluted cDNA sample is required for quality control using the miScript miRNA QC PCR Array cDNA prepared using the miScript Il RT Kit with miScript HiSpec Buffer is the appropriate starting material for this protocol This protocol describes quality control of multiple cDNA samples using the miScript SYBR Green PCR Kit and miScript miRNA QC PCR Array prior to miRNA profiling using miScript miRNA PCR Arrays In total 32 cDNA samples can be analyzed on one 384 well miScript miRNA QC PCR Array 8 cDNA samples can be analyzed on one 96 well miScript miRNA QC PCR Array and 8 cDNA samples can be analyzed on one Rotor Disc 100 miScript miRNA QC PCR Array If cDNA was preamplified using the miScript PreAMP PCR Kit with a miScript PreAMP Primer Mix refer to the miScript PreAMP Handbook for a preamplification speci
18. reverse transcription performance during profiling experiments with miScript miRNA PCR Arrays Two buffers 5x miScript HiSpec Buffer and 5x miScript HiFlex Buffer are provided in the miScript Il RT Kit to meet the distinctive needs of miRNA quantification studies using real time PCR Figure 1 The unique patent pending formulation of 5x miScript HiSpec Buffer facilitates the selective conversion of mature miRNAs into cDNA which can then be used for miRNA quantification with either miScript miRNA PCR Arrays or miScript Primer Assays Using miScript HiSpec Buffer the conversion of long RNAs such as mRNAs is suppressed As a result background signals potentially contributed by long RNAs are nonexistent making a reverse transcription reaction buffered using miScript HiSpec Buffer highly suitable for profiling the expression of mature miRNAs Only 5x miScript HiSpec Buffer should be used to prepare cDNA for real time PCR with miScript miRNA PCR Arrays For protocols using miScript Primer Assays refer to the miScript PCR System Handbook If working with samples that contain low amounts of RNA 5x miScript HiSpec Buffer should also be used to prepare cDNA for preamplification with the miScript PreAMP PCR Kit For protocols using the miScript PreAMP PCR Kit refer to the miScript PreAMP Handbook 5x miScript HiFlex Buffer promotes conversion of all RNA species mature miRNA precursor miRNA other noncoding RNA and mRNA into cDNA and this cDNA c
19. x 384 well plate or 8 x 96 well plates Rotor Discs add 200 RNase free water to the 20 reverse transcription reaction For 3 x 384 well plate or 12 x 96 well plates Rotor Discs add 310 ul RNase free water to the 20 ul reverse transcription reaction For 4 x 384 well plate or 16 x 96 well plates Rotor Discs add 420 RNase free water to the 20 reverse transcription reaction miScript miRNA PCR Array Handbook 05 2012 29 Protocol Real Time PCR for Mature miRNA Expression Profiling cDNA prepared using the miScript II RT Kit with miScript HiSpec Buffer with optional preamplification if required using the miScript PreAMP PCR Kit is the appropriate starting material for this protocol This protocol enables real time PCR profiling of mature miRNA using miScript miRNA PCR Arrays in combination with the miScript SYBR Green PCR Kit which contains the miScript Universal Primer reverse primer and QuantiTect SYBR Green PCR Master Mix Important points before starting The PCR must start with an initial incubation step of 15 minutes at 95 C to activate HotStarTaq DNA Polymerase included in 2x QuantiTect SYBR Green PCR Master Mix IMPORTANT Only miScript HiSpec Buffer should be used to prepare cDNA for real time PCR with miScript miRNA PCR Arrays IMPORTANT Do not use miScript HiFlex Buffer to prepare cDNA for real time PCR with miScript miRNA PCR Arrays Ensure that the 20 ul cDNA synthesis reac
20. 1 If all criteria described above are met cDNA samples are of sufficient quality for analysis Proceed with miScript miRNA PCR Array experiments miScript miRNA PCR Array Handbook 05 2012 45 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions Evidence of poor reverse transcription efficiency value of AVG C TC AVG gt 7 a Poor quality RNA Check the A260 A280 and ratios of the RNA samples Be sure to perform the dilutions for spectrophotometry in RNase free 10 mM Tris Cl pH 7 5 If necessary repurify RNA with a spin column based clean up method such as the miRNeasy Mini Kit cat no 217004 b Calculation did not If using miRNome miScript miRNA PCR Arrays include correction miScript miRNA HC PCR Arrays or miScript factor miRNA QC PCR Arrays be sure to include the correction factors detailed in Table 9 page 37 or Table 12 page 44 Evidence of poor overall PCR amplification efficiency AVG varies by more than 2 across arrays
21. 10 of each row contain replicate miRTC miScript Primer Assays miRTC Wells 11 and 12 of each row contain replicate positive PCR controls PPC These formats enable the quality assessment of upto 8 cDNA samples miScript miRNA PCR Array Handbook 05 2012 19 N 13 14 on 16 17 18 19 20 21 22 23 24 amp lt lt lt lt BOSS s L3 4 J PN 3 A 8 do EMI Y Da x OG DIO a OO om OX J y a C amp a E 2 a VO NI D A G a Y DG V EATE LEDIN AN G BN D DG D gt D gt A 2 ED A E 8 o 6966e6060666066060606 0000000000000000 0000000000000000 9 999299929924929299999 LANE ILL LL eee Re RR eeeese eee es ee ee ee 0S 06068660 2 do 8 Zz as amp 5898 eee elegans miScript snoRNA miScript miRTC Positive miScript Primer Assays PCR Con
22. 2 25 4 and 45 for the Silver Thermoblock adjust the ramp rate to 2696 for the Aluminum Thermoblock adjust the ramp rate to 3596 f using a Roche LightCycler 480 adjust the ramp rate to 1 C s 3 Due to software requirements the fluorescence detection step must be at least 30 s with the ABI PRISM 7000 or 34 s with the Applied Biosystems 7300 and 7500 If using a Roche LightCycler 480 use 45 cycles 8 Place the plate Rotor Disc in the real time cycler and start the cycling program 9 Perform data analysis 34 miScript miRNA PCR Array Handbook 05 2012 Protocol Data Analysis Using the AAC Method of Relative Quantification for miScript miRNA PCR Arrays This protocol describes the steps for analysis of data from miScript miRNA PCR Arrays The first steps should be performed by the user The later steps are performed by the free data analysis software This data analysis software for miScript miRNA PCR Arrays is available at http pcrdataanalysis sabiosciences com mirna Either the miScript miRNA PCR Array Web based software or the miScript miRNA PCR Array Data Analysis Excel Template can be accessed Both tools automatically perform quantification using the AAC method of relative quantification and interpretation of the control wells Results are presented in a tabular format a scatter plot a three dimensional profile and a volcano plot when replicates are included If cDNA was preamplified using the miScript PreAM
23. 4 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 174 176 177 178 179 180 181 182 183 184 185 1 8 8 8 5 8 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 20 211 212 213 214 215 216 217 218 219 220 201 222 223 204 225 226 227 208 209 230 231 232 233 234 235 236 237 238 239 240 24 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 323 324 325 326 327 328 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 c 91 90 99 90 95 88 C elegans snoRNA snRNA Reverse Positive miR 39 miScript miScript transcription PCR Primer Assay PCR Controls control control Figure 5 miRNome miScript miRNA PCR Array and miScript miRNA HC PCR Array layout for plate formats E G Wells A1 to P12 1 372 each contain a miScript Primer Assay for a miRNome or pathway disease functionally related mature miRNA Wells P13 and P14 contain replicate C elegans miR 39 miScript Primer Assays that can be used as an alternative normalizer for array data Ce Wells P15 to P20
24. 54 miScript miRNA PCR Array Handbook 05 2012 DNA contamination in RNA samples purified from different tissues Depending on the type of tissue used as starting material for RNA purification a fluorescent signal may also be generated in no RT control reactions When RNA is purified from tissues that contain large amounts of DNA such as spleen or thymus the level of DNase treatment required may be higher than for other tissues For such tissues we recommend performing a DNase digestion using the QIAGEN RNase Free DNase Set cat no 79254 when using the miRNeasy Mini and miRNeasy 96 Kits for RNA purification a miScript miRNA PCR Array Handbook 05 2012 55 Ordering Information Product miScript Il RT Kit 12 miScript Il RT Kit 50 miScript SYBR Green PCR Kit 200 miScript SYBR Green PCR Kit 1000 miScript Primer Assay 100 Pathway Focused miScript miRNA PCR Array miScript miRNA HC PCR Array miRNome miScript miRNA PCR Array Contents For 12 cDNA synthesis reactions miScript Reverse Transcriptase Mix 10x miScript Nucleics Mix 5x miScript HiSpec Buffer 5x miScript HiFlex Buffer RNase Free Water For 50 cDNA synthesis reactions miScript Reverse Transcriptase Mix 10x miScript Nucleics Mix 5x miScript HiSpec Buffer 5x miScript HiFlex Buffer RNase Free Water For 200 reactions QuantiTect SYBR Green PCR Master Mix miScript Universal Primer For 1000 reactions QuantiTect SYBR Green PCR Mas
25. 85 90 95 Figure 14 miRNA dissociation curve Dissociation curve analysis of an miR 21 PCR produd showing a single peak from the specific amplification product Dissociation curve analysis was performed using the Rotor Gene Q miScript miRNA PCR Array Handbook 05 2012 51 Appendix B General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient to degrade RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure In order to create and maintain an RNase free environment the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep purified RNA on ice when aliquots are pipett
26. A enriched fraction E Set up all reactions on to minimize the risk of RNA degradation Do not vortex template RNA or any of the components of the miScript RT Kit 26 miScript miRNA PCR Array Handbook 05 2012 Table 3 Recommended RNA starting amounts and buffers for reverse transcription reactions PCR application Assay Buffer Recommended RNA input Pathway profiling Pathway Focused 5x miScript 125 250 ng per of mature miRNA miScript miRNA HiSpec Buffer RNA sample PCR Arrays High content miScript miRNA HC 5x miScript 250 500 ng per pathway profiling PCR Arrays HiSpec Buffer RNA samplet of mature miRNA Whole miRNome miRNome miScript 5 miScript 250 500 ng per profiling of miRNA PCR Arrays HiSpec Buffer 384 plate or per mature miRNA 4 x 96 well plates Rotor Discs the number of plates provided in a miRNome miScript miRNA PCR Array varies depending on the species of interest If the RNA sample is not limiting use the upper amount of the recommended range t If RNA was prepared from 100 200 ul serum or plasma using the miRNeasy Serum Plasma Kit we recommend using 1 5 ul RNA prep approximately one tenth final eluate per miScript miRNA PCR Array If RNA was prepared from 100 200 ul serum or plasma using the miRNeasy Serum Plasma Kit we recommend using 1 5 ul RNA prep approximately one tenth final eluate per 384 well plate or per 4 x 96 well plates Rotor Discs 3 Depending on the RNA star
27. A free Web based miScript miRNA PCR Array data analysis tool simplifies the analysis of real time PCR data Once raw threshold cycle C data has been uploaded the tool automatically performs all fold change calculations using the AAC method of relative quantification and presents the results in several formats Mature miRNome expression profiling is now within reach of every laboratory because of the ease convenience and consistent performance of miScript miRNA PCR Arrays miScript miRNA PCR Arrays are at the forefront of real time PCR based mature miRNA profiling tools miScript miRNA PCR Array Handbook 05 2012 9 miScript miRNA PCR Array workflow Prepare reverse transcription reaction Incubate at 37 C for 60 min then at 95 C for 5 min Prepare PCR mix Add PCR mix to miScript miRNA PCR Array p Value for Fold Change 8 5 Normal Breast 5 4 3 2 1 3 4 0 1 2 Fold Change Ratio log 10 miScript miRNA PCR Array Handbook 05 2012 miScript II RT Kit The expanded miScript II RT Kit includes miScript Reverse Transcriptase Mix 10x miScript Nucleics Mix 5x miScript HiSpec Buffer and 5x miScript HiFlex Buffer miScript Reverse Transcriptase Mix is an optimized blend of poly A polymerase and reverse transcriptase 10x miScript Nucleics Mix contains dNTPs rATP oligo dT primers and an internal synthetic RNA control miRNA reverse transcription control miRTC that is used to assess
28. For protocols using miScript HiFlex Buffer refer to the miScript PCR System Handbook Only 5x miScript HiSpec Buffer should be used to prepare cDNA for real time PCR with miScript miRNA PCR Arrays Principle and procedure Mature miRNAs are naturally occurring 22 nucleotide noncoding RNAs that mediate posttranscriptional gene regulation Unlike mRNAs miRNAs are not polyadenylated in nature Reverse transcription using miScript HiSpec Buffer When reverse transcription reactions are performed in miScript HiSpec Buffer mature miRNAs and certain small nucleolar RNAs and small nuclear RNAs snoRNAs and snRNAs see Controls in miScript miRNA PCR Arrays and miScript miRNA QC PCR Arrays page 21 are selectively converted into cDNA The conversion of long RNAs such as mRNAs is suppressed Mature miRNAs 12 miScript miRNA PCR Array Handbook 05 2012 are polyadenylated by poly A polymerase and reverse transcribed into cDNA using oligo dT primers Figure 2 Polyadenylation and reverse transcription are performed in parallel in the same tube The oligo dT primers have a 3 degenerate anchor and a universal tag sequence on the 5 end allowing amplification of mature miRNA in the real time PCR step miScript miRNA PCR Arrays used in combination with the miScript SYBR Green PCR Kit enable quantification of mature miRNA by real time PCR The combination of polyadenylation and the universal tag addition ensures that miScript miRNA PCR Arrays do
29. May 2012 miScript miRNA PCR Array Handbook miScript Il RT Kit miScript SYBR Green PCR Kit miScript miRNA PCR Arrays miScript miRNA QC PCR Array For SYBR Green based real time PCR profiling of microRNAs using pathway focused arrays HC arrays and miRNome arrays QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www qiagen com Contents Kit Contents 4 Shipping and Storage 7 Intended Use 7 Safety Information 8 Quality Control 8 Introduction 9 Principle and procedure 12 Template RNA requirements 24 Equipment and Reagents to Be Supplied by User 25 Protocols B Reverse Transcription for Quantitative Real Time PCR 26 Real Time PCR for Mature miRNA Expression Profiling 30 m Data Analysis Using the AAC Method of Relative Quantification for miScript miRNA PCR Arrays 35 cDNA Quality Control Prior to Profiling Mature miRNA 39 Data Analysis for Quality Control Us
30. P PCR Kit with a miScript PreAMP Primer Mix refer to the miScript PreAMP Handbook for a data analysis protocol Important point before starting Text marked with a denotes instructions for 96 well and 384 well plates formats A C D and text marked with a A denotes instructions for 100 well Rotor Discs format R Procedure Steps performed by the user 1 Define the baseline The baseline is the noise level in early cycles where there is no detectable increase in fluorescence due to PCR products Use the Linear View of the amplification plot to determine the earliest visible amplification Set the baseline from cycle 2 to 2 cycles before the earliest visible amplification Do not use greater than cycle 15 The number of cycles used to calculate the baseline can be changed and should be reduced if high template amounts are used For more information regarding real time PCR data output refer to Appendix A page 49 For the Rotor Gene Q we recommend using the Dynamic Tube setting along with the Slope Correct and or Ignore First settings For more information refer to the Rotor Gene Q User Manual Note Ensure that baseline settings are the same across all PCR runs associated with the same experiment to allow comparison of results miScript miRNA PCR Array Handbook 05 2012 35 2 Define the threshold The threshold should be set using a logarithmic amplification plot so that the log linear
31. Pathway Focused miScript miRNA PCR Arrays plate format E G 384 well Add 10 ul reaction mix for each of 4 samples into the staggered wells with the same number as indicated in the figure miScript miRNA PCR Array Handbook 05 2012 33 5 Carefully tightly seal the miScript miRNA PCR Array with Optical Thin Wall 8 Cap Strips Formats A and D Optical Adhesive Film Formats C E F and G or Rotor Disc Heat Sealing Film Format R 6 Centrifuge the PCR plate for 1 min at 1000 g at room temperature 15 25 C to remove bubbles Note This step is not necessary for reactions set up in Rotor Discs 7 Program the real time cycler according to Table 8 Note Perform dissociation curve analysis of the PCR product s to verify their specificity and identity Dissociation curve analysis is an analysis step built into the software of real time cyclers Follow the instructions provided by the supplier Table 8 Cycling conditions for real time PCR Step Time Temperature Additional comments PCR 15 min 95 HotStarTag DNA Polymerase is Initial activated by this heating step activation step 3 step Denaturation 155 Annealing 30 s Extension 30 s Perform fluorescence data collection Cycle number 40 cycles Cycle number depends on the amount of template cDNA and abundance of the target For Bio Rad models CFX96 and CFX384 adjust the ramp rate to 1 C s t For Eppendorf Mastercycler ep realplex models
32. The miRTC miScript Primer Assay is an assay that assesses the performance of a reverse transcription reaction using the miScript Il RT Kit by detecting template synthesized from the kit s built in miRNA reverse transcription control RNA miRTC This control monitors for any variables that may inhibit the reverse transcription reaction The positive PCR control PPC wells contain a predispensed artificial DNA sequence and the assay that detects it This control monitors for any variables that may inhibit the PCR reaction Data analysis Free data analysis software for each miScript miRNA PCR Array is available at http pcrdataanalysis sabiosciences com mirna At this Web page both the miScript miRNA PCR Array Web based software and the miScript miRNA PCR Array Data Analysis Excel Template can be accessed Both tools will automatically perform quantification using the AAC method of relative quantification and interpretation of the control assays for more details see page 35 Results are presented in a tabular format a scatter plot a three dimensional profile and a volcano plot when replicates are included miScript miRNA PCR Array Handbook 05 2012 23 Template RNA requirements Total RNA containing miRNA is the required starting material for miScript miRNA PCR Arrays It is not necessary to enrich for small RNA QIAGEN provides a range of solutions for purification of total RNA including miRNA Table 2 Table 2 Kits for pu
33. all species currently supported by miScript miRNA PCR Arrays These benefits make the miScript Primer Assays for miR 16 miR 21 and miR 191 ideal for sample quality assessment For accurate and reproducible results in miRNA quantification by real time PCR if is necessary to normalize the amount of target miRNA by using a suitable endogenous reference RNA This approach is known as relative quantification Normalization corrects for factors that could otherwise lead to inaccurate quantification These factors include variation in quantity of input RNA possible RNA degradation or presence of inhibitors in the RNA samples and differences in sample handling Normalization also allows results from different experiments and samples to be compared directly miScript PCR Controls are primers designed to quantify a panel of 5 snoRNAs SNORD61 SNORD68 SNORD72 SNORD95 and SNORD96A and the snRNA RNU6B RNU6 2 These controls take into consideration sequence homologies in human mouse rat dog and rhesus macaque so that the same controls can be used for all 4 species In addition these small RNAs have been verified to have relatively stable expression levels across tissues and cell types As a result miScript PCR Controls serve as normalization controls for relative quantification using the miScript PCR System All the controls have amplification efficiencies close to 100 For more information and to view data visit www giagen com miRNAControls
34. an then be used to quantify each RNA species using appropriate primer assays Do not use 5x miScript HiFlex Buffer to prepare cDNA for real time PCR with miScript miRNA PCR Arrays For protocols using miScript HiFlex Buffer refer to the miScript PCR System Handbook miScript miRNA PCR Array Handbook 05 2012 11 miScript Il RT Kit cDNA synthesis cDNA synthesis using miScript using miScript HiSpec Buffer HiFlex Buffer Realtime PCR Realtime PCR profiling quantification quantification of mature of mature miRNA only miRNA precursor miRNA ncRNA and or mRNA Figure 1 Mature miRNA precursor miRNA other noncoding RNA and mRNA detection Two buffers are provided with the miScript II RT Kit Use miScript HiSpec Buffer for cDNA synthesis to enable either mature miRNA profiling Using miScript miRNA PCR Arrays or mature miRNA quantification using individual miScript Primer Assays For protocols using miScript HiSpec Buffer in combination with miScript Primer Assays refer to the miScript PCR System Handbook If samples being assessed have low RNA content miScript HiSpec Buffer should be used to prepare cDNA for preamplification with the miScript PreAMP PCR Kit For protocols using the miScript PreAMP PCR Kit refer to the miScript PreAMP Handbook Use miScript HiFlex Buffer for cDNA synthesis to enable quantification of mature miRNA precursor miRNA other noncoding RNA ncRNA and or mRNA from the same cDNA using appropriate primer assays
35. ations outside the United States owned by Roche Diagnostics GmbH for internal research use or for non in vitro diagnostics applications with authorized reagents with regard to Melting Curve Analysis No right is conveyed expressly by implication or estoppel under any other patent or patent claims owned by Roche Diagnostics GmbH or by any other Party NOTICE TO PURCHASER DISCLAIMER OF LICENSE No license is conveyed with the purchase of this product miScript Reverse Transcription Kit miScript Primer Assay miScript Precursor Assay under any of US Patents Nos 5 804 375 5 994 056 6 171 785 5 538 848 5 723 591 5 876 930 6 030 787 and 6 258 569 and corresponding paini outside the United States or any other patents or patent applications relating to the 5 Nuclease and dsDNA Binding Dye Processes For urther information contact the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City Cdlifornia 94404 USA NOTICE TO PURCHASER LIMITED LICENSE The use of this product miScript SYBR Green PCR Kit is covered by at least one claim of U S Patent No 7 687 247 owned by Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable rightto use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components
36. d HotStarTag DNA Ensure that the cycling program includes the hot Polymerase not start activation step for HotStarTaq DNA activated with a hot polymerase for details check the protocol start e No detection activated Check that fluorescence detection was activated in the cycling program f Wrong detection step Ensure that fluorescence detection takes place during the extension step of the PCR cycling program g Wrong dye layer filter Ensure that the appropriate layer filter is chosen activated h Insufficient starting Increase the amount of template cDNA template No linearity in ratio of value crossing point to log of the template amount a Template amount too Do not exceed maximum recommended high amounts of template cDNA For details see the protocol b Template amount too Increase amount of template RNA low Varying fluorescence intensity a Real time cycler Decontaminate the real time cycler according to contaminated the supplier s instructions b Real time cycler no Recalibrate the real time cycler according to the longer calibrated supplier s instructions 48 miScript miRNA PCR Array Handbook 05 2012 Appendix A Real Time PCR Data Output and Dissociation Curve Analysis In a typical amplification plot resulting from a real time PCR reaction fluorescence is plotted against the number of cycles producing sigmoidal shaped plots when using a linear scale The threshold cycle C serves as a tool fo
37. ed for downstream applications To remove RNase contamination from bench surfaces nondisposable plasticware and laboratory equipment e g pipets and electrophoresis tanks use of RNasekKiller cat no 2500080 from 5 PRIME www 5prime com is recommended RNase contamination can alternatively be removed using general laboratory reagents To decontaminate plasticware rinse with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 53 or rinse with chloroform if the plasticware is chloroform resistant To decontaminate electrophoresis tanks clean with detergent e g 0 596 505 rinse with RNase free water rinse with ethanol if the tanks are ethanol resistant and allow to dry Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally RNase free and do not require pretreatment to inactivate RNases When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material data sheets MSDSs available from the product supplier 52 miScript miRNA PCR Array Handbook 05 2012 Glassware Glassware should be treated before use to ensure that it is RNase free Glassware used for RNA work should be cleaned with a detergent thoroughly rinsed and oven baked at 240 C for 4 hours or more overnight if more convenient before use Autoclaving a
38. eline The baseline is the noise level in early cycles where there is no detectable increase in fluorescence due to PCR products Use the Linear View of the amplification plot to determine the earliest visible amplification Set the baseline from cycle 2 to 2 cycles before the earliest visible amplification Do not use greater than cycle 15 The number of cycles used to calculate the baseline can be changed and should be reduced if high template amounts are used For more information regarding real time PCR data output refer to Appendix A page 49 For the Rotor Gene Q we recommend using the Dynamic Tube setting along with the Slope Correct and or Ignore First settings For more information refer to the Rotor Gene Q User Manual Note Ensure that baseline settings are the same across all PCR runs in the same analysis to allow comparison of results 2 Define the threshold The threshold should be set using a logarithmic amplification plot so that the log linear range of the curve can be easily identified Using the Log View of the amplification plot place the threshold above the background signal but within the lower half of the log linear range of the amplification plot The threshold should never be set in the plateau phase The absolute 42 miScript miRNA PCR Array Handbook 05 2012 position of the threshold is less critical than its consistent position across PCR runs Various PCR instruments such as Applied Bi
39. ent Technologies Bio Rad iCycler Chromo4 CFX96 DNA Engine Opticon CFX384 iQ MyiQ Bio Rad Laboratories Inc Excel Microsoft Inc Registered names trademarks etc used in this document even whennot specifically marked as such are not to be considered unprotected by law Use of this product miScript SYBR Green PCR Kit is covered by one or more of the following US patents and corresponding patent claims outside the US 5 994 056 and 6 171 785 The purchase of this product includes a limited nontransferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA The purchase of this product miScript SYBR Green PCR Kit includes a limited non transferable license under U S Patent No 5 871 908 and all continuations and divisionals and corresponding claims in patents and patent applic
40. f Be sure to set up the reaction on ice reverse transcription reaction c Poor quality or Check the concentration integrity and purity of incorrect amount of the template RNA before starting the protocol template RNA for Mix well after thawing the template RNA Even reverse transcription minute amounts of RNases can affect synthesis of reaction cDNA and sensitivity in RT PCR particularly with small amounts of RNA d RNA concentration too See Table 3 page 27 for recommended RNA high or too low amounts e RNA denatured Denaturation of the template RNA is not necessary If denaturation was performed the integrity of the RNA may be affected f Incubation temperature Reverse transcription should be carried out at too high 37 Higher temperatures may reduce the length of cDNA products or the activity of miScript Reverse Transcriptase Mix Check the temperature of your heating block or water bath miScript miRNA PCR Array Handbook 05 2012 47 Comments and suggestions No product or product detected late in real time PCR or only primer dimers detected indicative of problems occurring during real time PCR a PCR annealing time too the annealing time specified in the protocol short b PCR extension time too Use the extension time specified in the protocol short Pipetting error Check the concentrations and storage conditions missing reagent when of reagents including primers and cDNA setting up PCR
41. fic quality control protocol Important points before starting The must start with an initial incubation step of 15 minutes at 95 C to activate HotStarTaq DNA Polymerase included in 2x QuantiTect SYBR Green PCR Master Mix Only cDNA template prepared using miScript HiSpec Buffer should be used with this protocol E Ensure cDNA samples were prepared according to the protocol on page 26 and that the 20 ul cDNA synthesis reaction has been diluted appropriately see Table 5 page 29 Do not vortex template cDNA or any of the components of the miScript SYBR Green PCR Kit The standard miScript miRNA PCR Array reaction volumes are 10 ul per well for a 384 well plate 25 ul per well for a 96 well plate and 20 ul per well for a 100 well Rotor Disc M If using the iCycler iQ iQ5 or MyiQ well factors must be collected at the beginning of each experiment Well factors are used to compensate for any system or pipetting nonuniformity For details refer to the user manual supplied with the instrument or Technical Information Using QuantiTect SYBR Green Kits on Bio Rad cyclers available at www giagen com miScript miRNA PCR Array Handbook 05 2012 39 Procedure 1 Thaw 2x QuantiTect SYBR Green PCR Master Mix 10x miScript Universal Primer template cDNA and RNase free water at room temperature 15 25 Mix the individual solutions 2 Prepare a reaction mix according to Table 10 Mix gently and thoroughly
42. fluorescence is high However at high temperatures PCR products are denatured resulting in rapid decreases in fluorescence The fluorescence is measured continuously as the temperature is increased and the fluorescence values are plotted against temperature A curve is produced because fluorescence decreases slightly through the lower end of the temperature range but decreases much more rapidly at higher temperatures as the dissociation temperatures of nonspecific and specific PCR products are reached The detection systems calculate the first derivatives of the curves resulting in curves with peaks at the respective Tms Figures 13 and 14 Curves with peaks at a T lower than that of the specific PCR product indicate the formation of primer dimers while diverse peaks with different T s or plateaus indicate production of nonspecific products or a smear Specific product sample 4 Primer dimer 4 Fluorescence dF dT al Sample B Temperature Figure 13 Dissociation curve analysis Dissociation curve analysis of 2 samples and B Sample A yields only 1 peak resulting from the specific amplification product primer dimers not coamplified Sample B shows a peak from the specific product and a peak at a lower temperature from amplification of primer dimers 50 miScript miRNA PCR Array Handbook 05 2012 60 65 70 75 80
43. iRNA PCR Arrays and miScript miRNA QC PCR Arrays are shipped at ambient temperature on ice or on dry ice depending on the destination and accompanying products Upon receipt store at 20 C If stored under these conditions miScript miRNA PCR Arrays and miScript miRNA QC PCR Arrays are stable for 6 months after receipt Intended Use The miScript Il RT Kit miScript SYBR Green PCR Kit miScript miRNA PCR Arrays and miScript miRNA QC PCR Array are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease miScript miRNA PCR Array Handbook 05 2012 7 All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www qiagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany
44. iling experiments The miScript PreAMP PCR Kit in combination with miScript PreAMP Primer Mixes uses highly multiplex PCR based preamplification of up to 400 miRNA specific cDNA targets in one reaction This breakthrough technology enables accurate and comprehensive miRNome wide expression analysis with as little as 10 ng total RNA For more information refer to the miScript PreAMP Handbook or visit www giagen com miRNA Mature miRNA expression profiling using miScript miRNA PCR Arrays cDNA prepared in a reverse transcription reaction using miScript HiSpec Buffer serves as the template for real time PCR analysis using a miScript miRNA PCR Array which contains miRNA specific miScript Primer Assays and the miScript SYBR Green Kit which contains the miScript Universal Primer reverse primer and QuantiTect SYBR Green PCR Master Mix To profile mature miRNA expression a premix of cDNA miScript Universal Primer QuantiTect SYBR Green PCR Master Mix and RNase free water is added to a miScript miRNA PCR Array miScript miRNA PCR Array plate layout miScript miRNA PCR Arrays are available in 96 well 384 well and Rotor Disc 100 formats Figures 3 6 Each array contains several control assays The purpose of each control is described on page 21 14 miScript miRNA PCR Array Handbook 05 2012 2 6 7 9 0 1L 12 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23
45. ing miScript miRNA QC PCR Arrays 42 Troubleshooting Guide 46 Appendix A Real Time PCR Data Output and Dissociation Curve Analysis 49 Appendix B General Remarks on Handling RNA 52 Appendix C Preparation Quantification and Storage of RNA 54 Ordering Information 56 ioo H Immm miScript miRNA PCR Array Handbook 05 2012 3 Kit Contents miScript II RT Kit 12 50 Catalog no 218160 218161 Number of standard reactions 12 50 miScript Reverse Transcriptase Mix 24 ul 100 ul 10x miScript Nucleics Mix 50 200 ul 5x miScript HiSpec Buffer 100 ul 400 5x miScript HiFlex Buffer 100 ul 400 RNase Free Water 1 9 ml 1 9 ml Quick Start Protocol 1 1 A standard reaction is 20 in volume with up to 2 ug total RNA when using miScript HiSpec Buffer miScript SYBR Green PCR Kit 200 1000 Catalog no 218073 218075 Number of 50 pl reactions 2001 1000 2x QuantiTect SYBR Green PCR Master 3x1 7 25 containing HotStarTaq DNA Polymerase QuantiTect SYBR Green PCR Buffer dNTP mix including dUTP SYBR Green ROX passive reference dye 5 mM MgCl 10x miScript Universal Primer 1 ml 5xl ml RNase Free Water 2x2ml 20 ml Quick Start Protocol 2 2 t Provides sufficient reagents for 4 x 96 well arrays 110 ul residual 2 x 384 well pathway focused arrays 1210 ul residual 2 x 384 well HC miRNome arrays 1510 residual or 5 Rotor Disc 100 arrays 110 ul residual Provides sufficie
46. is considered a negative call values of the positive PCR control wells PPC are examined If the RNA sample is of high quality the cycling program has been correctly run and the thresholds have been correctly defined the value of should be 19 2 A 15 2 across all arrays or samples C values of the reverse transcription control miRTC are examined using the values for the positive PCR control PPC by calculating AC AVG AVG C PPC If this value is less than 7 then no inhibition of the reverse transcription reaction is apparent No action is needed If this value is greater than 7 there is evidence of impurities that may have inhibited the reverse transcription reaction See the Troubleshooting Guide page 46 miScript miRNA PCR Array Handbook 05 2012 Note The AVG AVG CS calculation is specific for Pathway Focused miScript miRNA PCR Arrays For miRNome miScript miRNA PCR Arrays as the cDNA is divided among significantly more wells a correction factor is introduced into the calculation according to the number of plates Rotor Discs that are being used This corrects for the dilution of the miRTC The calculation is performed as shown in Table 9 Table 9 AC FTC calculation for miRNome miScript miRNA PCR Arrays and miScript miRNA HC PCR Arrays Number of plates Rotor Discs 384 96 Rotor Correction calculation well well Disc 100 factor
47. ity of multiple cDNA samples using real time PCR miScript miRNA QC PCR Arrays are available in 96 well 384 well and Rotor Disc 100 formats Figures 7 9 The purpose of each control is described on page 21 The 96 well plate and Rotor Disc 100 formats allow quality control of up to 8 cDNA samples The 384 well format allows quality control of up to 32 cDNA samples 18 miScript miRNA PCR Array Handbook 05 2012 9 miRTC EY lt gt amp uw lt w diii mirc lt miRTC p PPC Ss 4 2 2 PPC PPC e A Vay Se C elegans miScript snoRNA miRTC Positive miScript Primer Assays miScript PCR miScript PCR Primer Assay Controls Primer Assay control rm mn me e 0000000 0000000 9000000 0000000 0000000 Figure 7 miScript miRNA QC PCR Array layout for plate formats A C D Wells 1 and 2 of each row contain replicate C elegans miR 39 miScript Primer Assays 39 Well 3 of each row contains a miR 16 miScript Primer Assay 16 Well 4 of each row contains a miR 21 miScript Primer Assay 21 Well 5 of each row contains a miR 191 miScript Primer Assay 191 Wells 6 to 8 of each row each contain an assay for a different snoRNA 61 SNORD641 assay 95 SNORD95 assay 96 SNORD96A assay Wells 9 and
48. l and 96 well array formats a multichannel pipettor can be used to add reaction mix to the array For the Rotor Disc 100 format a repeater pipettor or a GllAgility can be used to load the array For 384 well miScript miRNA PCR Array add 10 ul per well For 96 well miScript miRNA PCR Array add 25 ul per well For Rotor Disc miScript miRNA PCR Array add 20 ul per well Loading 384 well 4 x 96 Pathway Focused miScript miRNA PCR Arrays Note Each Pathway Focused miScript miRNA PCR Array contains 4 replicates of 96 assays that can be used for analysis of 4 samples The 32 miScript miRNA PCR Array Handbook 05 2012 spacing between the tips of standard multichannel pipettors allows rows or columns to be skipped when adding each sample Be sure to load each sample into the correct set of wells using a multichannel pipettor and the 384EZLoad Covers provided Use Figure 10 as a guide Do not reuse 384EZLoad Covers Place 384EZLoad Cover 1 white the plate Add 10 ul reaction mix for sample 1 to the open wells odd number wells of rows A C E K M and Remove and discard 384EZLoad Cover 1 Place 384EZLoad Cover 2 yellow on the plate Add 10 reaction mix for sample 2 to the open wells even number wells of rows A C E M and Remove and discard 384EZLoad Cover 2 Place 384EZLoad Cover 3 black on the plate Add 10 ul reaction mix for sample 3 to the open wells odd number wells of
49. ld fall between 0 15 and 1 0 Note that absorbance measurements cannot discriminate between DNA and RNA Depending on the method used for RNA preparation RNA may be contaminated with DNA and this will result in misleadingly high Azo values The ratios between the absorbance values at 260 nm and 280 nm and at 260 nm and 230 nm give an estimate of RNA purity To determine RNA purity we recommend measuring absorbance 10 mM Tris Cl pH 7 5 Pure RNA has an ratio of 1 9 2 1 and an As so A 3o ratio of 2 0 2 2 Lower ratios indicate the presence of contaminants such as proteins Storage of RNA Purified RNA should be stored at 20 C 70 C in RNase free water When purified using QIAGEN systems no degradation is detectable for at least 1 year under these conditions Diluted solutions of RNA e g dilution series used as standards should be stored in aliquots and thawed once only We recommend storage of aliquots in siliconized tubes where possible This avoids adsorption of the RNA to the tube walls which would reduce the concentration of in solution When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Values up to 2 3 are routinely obtained for pure RNA in 10 mM Tris Cl pH 7 5 with some spectrophotometers
50. lone will not fully inactivate many RNases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate as described in Solutions below Solutions Note QIAGEN solutions such as miScript Nucleics Mix miScript HiFlex Buffer miScript HiSpec Buffer and RNase free water are guaranteed RNase free without using DEPC treatment and are therefore free of any DEPC contamination Solutions water and other solutions should be treated with 0 196 DEPC DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 196 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC will react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and When preparing Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affected u
51. miScript miRNA QC PCR Array t Only included Pathway Focused miScript miRNA PCR Array miRNome miScript miRNA PCR Array and miScript miRNA HC PCR Array The miScript Primer Assay for C elegans miR 39 detects the miRNeasy Serum Plasma Spike In Control cat no 219610 also called Syn cel miR 39 miScript miRNA Mimic cat MSYOOO00010 which is a C elegans miR 39 mimic This mimic can be added to samples particularly serum or plasma samples to control for variations during the preparation of total RNA and subsequent steps After purification real time RT PCR detection of the miRNeasy Serum Plasma Spike In Control can be performed and these results can then be used for normalization of real time RT PCR results for endogenous miRNAs in the sample For more information on the use of the miRNeasy Serum Plasma Spike In Control see the miRNeasy Serum Plasma Handbook The miScript Primer Assays for miR 16 miR 21 and miR 191 are included in the miScript miRNA QC PCR Array since their targeted miRNAs are ubiquitously expressed in a wide range of cells tissues and body fluid samples including whole blood serum and plasma This is in contrast to the snoRNA snRNA miScript PCR Controls whose intended targets are typically only expressed in 22 miScript miRNA PCR Array Handbook 05 2012 cells tissues and whole blood samples In addition the sequences for these mature miRNAs are conserved across a wide range of species including
52. nless a large fraction of the purine residues have been modified Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100 C for 15 minutes When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material data sheets MSDSs available from the product supplier miScript miRNA PCR Array Handbook 05 2012 53 Appendix C Preparation Quantification and Storage of RNA RNA preparation and quality Since PCR consists of multiple rounds of enzymatic reactions it is more sensitive to impurities such as proteins phenol chloroform salts and EDTA than single step enzyme catalyzed reactions Purity of nucleic acid templates is particularly important for real time PCR since contaminants can interfere with fluorescence detection See Table 2 page 24 for kits recommended for the purification of total RNA that includes miRNA For more information about kits for miRNA purification visit www qiagen com miRNA Determining concentration and purity of RNA The concentration of RNA should be determined by measuring the absorbance at 260 nm in spectrophotometer The sample should be diluted in water since the relationship between absorbance and concentration reading of 1 40 ug ml RNA is based on an extinction coefficient calculated for RNA in water To ensure significance readings shou
53. not detect genomic DNA 5 3 miRNA AAAA A 3 Polyadenylation 9 AAAA A 3 Reverse transcription pata HT 5 using miScript Oligo dT primer HiSpec Buffer with universal tag 5 AAAAIA 3 3 ee 5 5 Cole SLL LLIB Ist PCR cycle miScript Primer Assay miScript Primer Assay forward primer 3 mne RR 5 Subsequent miScript Universal Primer reverse primer Figure 2 Conversion of mature miRNAs into cDNA and subsequent detection In a reverse transcription reaction using miScript HiSpec Buffer mature miRNAs are polyadenylated by poly A polymerase and subsequently converted into cDNA by reverse transcriptase with oligo dT priming The cDNA is then used for real time PCR profiling of mature miRNA expression using a miScript miRNA PCR Array and the miScript Universal Primer miScript miRNA PCR Array Handbook 05 2012 13 Optional cDNA preamplification using miScript PreAMP PCR The miScript PreAMP PCR Kit enables researchers to perform miRNA profiling experiments using very limited amounts of starting RNA This is particularly important when working with samples such as body fluids formalin fixed paraffin embedded FFPE samples and small cell number samples such as laser capture microdissection LCM samples flow sorted cells circulating tumor cells and fine needle biopsies where the low RNA yields obtained are often insufficient for reliable miRNA prof
54. nt reagents for 20 x 96 well arrays 0 ul residual 12 x 384 well pathway focused arrays 1100 ul residual 13 x 384 well HC miRNome arrays 850 ul residual or 25 Rotor Disc 100 arrays 0 ul residual 4 miScript miRNA PCR Array Handbook 05 2012 Pathway Focused miScript miRNA PCR Array Catalog no Varies Format A C D 96 well plate containing 2 12 2 12 2 12 dried assays or 24 or24 or 24 384 well plate containing dried assays Rotor Disc 100 containing dried assays Optical Thin Wall 8 Cap 24 24 Strips 12 per plate 144 4 144 or or 288 288 Optical Adhesive Film 1 per plate 384EZLoad Covers 1 set of 4 per plate Rotor Disc Heat Sealing Film 1 per Rotor Disc miScript miRNA HC PCR Array Catalog no Varies Format E G 384 well plate containing dried 2 12 24 2 12 or 24 assays Optical Adhesive Film 1 per plate 2 12 or 24 2 12 or 24 miScript miRNA PCR Array Handbook 05 2012 5 miRNome miScript miRNA PCR Array Catalog no Varies Format A C D F G 36 well plate containing Varies Varies Varies Varies dried assays 384 well plate Varies Varies containing dried assays Rotor Disc 100 containing dried assays Varies Optical Thin Wall 8 Cap VOR Strips 12 per plate Optical Adhesive Film 1 Varies Varies Varies per plate Rotor Disc Heat Sealing Film 1 per Rotor Disc Meu miScript miRNA QC PCR Array Catalog n
55. o Varies Format A 96 well plate containing 1 dried assays 384 well plate containing dried assays Rotor Disc 100 containing dried assays Optical Thin Wall 8 Cap Strips Optical Adhesive Film Rotor Disc Heat Sealing Film 1 per Rotor Disc 6 miScript miRNA PCR Array Handbook 05 2012 Cyclers for use with array formats Format Suitable real time cyclers Plate Applied Biosystems models 5700 7000 7300 7500 gt 96 well 7700 7900HT ViiA 7 96 well block Bio Rad models iCycler iQ 5 MyiQ MyiQ2 Bio Rad MJ Research Chromo4 Eppendorf Mastercycler ep realplex models 2 2S 4 4S Stratagene models Mx3005P Mx3000P Takara TP 800 Applied Biosystems models 7500 Fast block 7200HT 96 well Fast block StepOnePlus 7 Fast block Bio Rad CFX96 Bio Rad MJ Research models DNA 96 well Engine Opticon DNA Engine Opticon 2 Stratagene Mx4000 Applied Biosystems models 7900HT 384 well block 384 well ViiA 7 384 well block Bio Rad CFX384 Roche LightCycler 480 96 well block 96 well Roche LightCycler 480 384 well block 384 well Rotor Gene Rotor Gene 6000 other Rotor Gene Rotor cyclers Disc 100 Shipping and Storage The miScript II RT Kit and miScript SYBR Green PCR Kit are shipped on dry ice The kits including all reagents and buffers should be stored immediately upon receipt at 20 in a constant temperature freezer miScript m
56. osystems models 7500 and VIIA 7 and Stratagene models Mx3005P and Mx3000P may require adjustment of the default Manual C threshold value of 0 2 to a lower value in order to analyze the data properly Use a value of 0 02 as a starting point For the Rotor Gene we recommend a C threshold value of approximately 0 02 in order to analyze the data properly Note Ensure that threshold settings are the same across all PCR runs in the same analysis to allow comparison of results 3 Export values according to the manual supplied with the real time PCR instrument 4 Access the free data analysis tools at http pcrdataanalysis sabiosciences com mirna Follow the instructions provided in the Excel template Steps performed by data analysis Excel template 5 All C values reported as greater than 35 or as N A not detected are changed to 35 or A as greater than 33 or as N A not detected are changed to 33 At this point any value equal to 35 or A 33 is considered a negative call 6 C values of the positive PCR control wells PPC are examined If the RNA sample is of high quality the cycling program has been correctly run and the thresholds have been correctly defined the value of should be 19 2 A 15 2 across all arrays or samples 7 C values of the reverse transcription control miRTC are examined using the values for the positive PCR control PPC by calculating AC AVG
57. per well miScript miRNA PCR Array Handbook 05 2012 31 Table 7 Reaction mix for miRNome miScript miRNA PCR Arrays and miScript miRNA HC PCR Arrays Array format 384 well 96 well Rotor Disc 100 Component Formats E G Formats A C D F Format R 2x QuantiTect 2050 1375 yl 1100 ul SYBR Green PCR Master Mix miScript 410 275 220 Universal Primer RNase free water 1540 1075 855 ul Template cDNA 100 ul 25 ul 25 ul Total volume 4100 pl 2750 yl 2200 yl Volumes are for a single plate or Rotor Disc associated with a miRNome set The number of plates in a miRNome set vary depending on the species Scale up volumes according to the number of plates Rotor Discs to be run If the miRNome set contains a plate Rotor Disc that is less than half full scale down volumes accordingly t miScript miRNA HC PCR Arrays use the volumes shown for 384 well Formats No optimization of the Mg concentration is required The final concentration provided by 2x QuantiTect SYBR Green PCR Master Mix gives optimal results Provides 0 5 1 ng cDNA per well 3 Carefully remove the miScript miRNA PCR Array from its sealed bag Optional for 96 well and 384 well array formats If the reaction mix is in a tube transfer to a loading reservoir such as the RT PCR Array Loading Reservoir cat no 338162 4 Add reaction mix to each well of the miScript miRNA PCR Array as follows Note For 384 wel
58. r calculation of the starting template amount in each sample This is the cycle in which there is the first detectable increase in fluorescence There may be variation in how determination of C values is carried out depending on the real time PCR cycler that is used Sample A log linear phase Fluorescence Earliest visible amplification Se 10 20 30 40 U 4 Cycle number Baseline Figure 11 Amplification plot Amplification plots showing increases in fluorescence from 2 samples A and B Sample A contains a higher amount of starting template than sample B 0 6 0 5 o E Norm Fluoro 5 01 oo 277 5 10 15 20 25 30 35 40 Cvcle Figure 12 Typical amplification plot Amplification plot after quantification of a range of amounts of miR 21 Real time PCR was performed using the Rotor Gene Q miScript miRNA PCR Array Handbook 05 2012 49 Dissociation curve analysis A dissociation curve analysis of PCR product s may be optionally performed to aid in verifying their specificity and identity Dissociation curve analysis is an analysis step built into the software of real time cyclers Follow instructions provided by the supplier To carry out dissociation curve analysis the temperature is increased very slowly from a low temperature e g 65 C to a high temperature e g 95 C At low temperatures PCR products are double stranded so SYBR Green dye binds to them and
59. range of the curve can be easily identified Using the Log View of the amplification plot place the threshold above the background signal but within the lower half of the log linear range of the amplification plot The threshold should never be set in the plateau phase The absolute position of the threshold is less critical than its consistent position across PCR runs Various PCR instruments such as Applied Biosystems models 7500 and VIIA 7 and Stratagene models Mx3005P and Mx3000P may require adjustment of the default Manual C threshold value of 0 2 to a lower value in order to analyze the data properly Use a value of 0 02 as a starting point For the Q we recommend a threshold value of approximately 0 02 in order to analyze the data properly Note Ensure that threshold settings are the same across all PCR runs in the same analysis to allow comparison of results Export C values according to the manual supplied with the real time PCR instrument Access the free data analysis tools at http pcrdataanalysis sabiosciences com mirna Choose either the Web based software or the Excel template and follow the instructions provided Steps performed by data analysis software 5 36 values reported as greater than 35 as N A not detected are changed to 35 or A as greater than 33 or as N A not detected are changed to 33 At this point any value equal to 35 or A 33
60. rification of RNA including miRNA Kit Cat no Starting material miRNeasy Micro Kit 217084 Small amounts of cells and tissues miRNeasy Mini Kit 217004 Animal human tissues and cells miRNeasy 96 Kit 217061 Animal human tissues and cells miRNeasy 217184 Animal and human plasma and Serum Plasma Kit serum miRNeasy FFPE Kit 217504 FFPE tissue samples PAXgene Tissue miRNA 766134 Animal human tissues that have Kit been fixed and stabilized in PAXgene Tissue Containers PAXgene Blood miRNA 763134 Human blood that has been Kit stabilized in PAXgene Blood RNA Tubes 24 miScript miRNA PCR Array Handbook 05 2012 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier For reverse transcription Thin walled DNase free RNase free PCR tubes for 20 ul reactions B Thermal cycler heating blocks or water baths capable of reaching 95 gm Microcentrifuge For quantitative real time PCR Real time PCR cycler the table on page 7 shows the appropriate real time cycler for each array format Multichannel pipettor Nuclease free pipet tips and tubes miScript miRNA PCR Array Handbook 05 2012 25 Protocol Reverse Transcription for Quantitative Real Time PCR Important points before sta
61. rows B D F H J L N and P Remove and discard 384EZLoad Cover 3 Place 384EZLoad Cover 4 red on the plate Add 10 ul reaction mix for sample 4 to the open wells even number wells of rows B D F H J L N and P Remove and discard 384EZLoad Cover 4 Cover 1 white for sample 1 Cover 2 yellow for sample 2 gt gt gt gt gt 2 0 0 0 0 0 0 0 0 0 0 43434 434 343434343 4 EBDEBBBEBSDBSBEBSHEBEBSHEU 2 gt gt gt BE TEBE naa aes ese 4 EIETEIESEREIEIETEIEYEIEIEIEJEIEIEI 2 gt gt 2 gt 2 gt gt gt 2 gt 2 gt gt gt 2 2 2 2 2 gt mg EICIEICIEIESEIEIEREIEIEREREREREIEIEREREREIEREIES EICIEIEEIEJEIEJEIEIEIEREIBIEIDIEIEIEIEXHIES 2 2 13434343434343434343434 UB TE EBEBHEHEBEHBBHEBEHEBEBEHB EBDEBBHEBESHBBEBSHBSBEBRSHER BD BD 0 0 og 2 2 4 gt gt gt gt 2 2 gt gt 2 gt 2 gt 2 gt gt
62. rting The miScript Il RT Kit includes 2 buffers 5x miScript HiSpec Buffer and 5x miScript HiFlex Buffer Only 5x miScript HiSpec Buffer should be used to prepare cDNA for mature miRNA profiling using Pathway Focused or miRNome miScript miRNA PCR Arrays or miScript miRNA HC PCR Arrays IMPORTANT Only miScript HiSpec Buffer should be used to prepare cDNA for real time PCR with miScript miRNA PCR Arrays IMPORTANT Do use miScript HiFlex Buffer to prepare cDNA for real time PCR with miScript miRNA PCR Arrays IMPORTANT If performing a reverse transcription reaction for use with the miScript PreAMP PCR Kit refer to the reverse transcription protocol in the miScript PreAMP Handbook Total RNA containing miRNA should be used as starting material For RNA purification recommendations see page 24 This protocol is for use with up to 2 ug quantifiable RNA or up to 9 ul RNA prepared from 100 200 serum or plasma using the miRNeasy Serum Plasma Kit If using higher RNA amounts scale up the reaction linearly Recommended starting amounts are shown in Table 3 If working with RNA for the first time read Appendix B page 52 if an miRNA enriched fraction has been prepared separate from larger RNAs e g using the miRNeasy Mini Kit use one tenth the recommended RNA input for total RNA For example if 125 250 ng total RNA is recommended for use with a Pathway Focused miScript miRNA PCR Array use 12 5 25 ng miRN
63. sma using the miRNeasy Serum Plasma Kit up to 9 ul RNA prep can be added to the reverse transcription reaction sufficient for 6 x 384 well plates or 24 x 96 well plates Rotor Discs 3 Add template RNA to each tube containing reverse transcription master mix Gently mix briefly centrifuge and then store on ice 4 Incubate for 60 min at 37 C 5 Incubate for 5 min at 95 C to inactivate miScript Reverse Transcriptase Mix and place on ice 6 Dilute the cDNA in RNase free water according to Table 5 and proceed with real time PCR immediately If you wish to store the reverse transcription reactions prior to real time PCR transfer the undiluted cDNA to 20 C freezer or dispense the diluted cDNA into 110 ul aliquots and transfer them to a 20 C freezer 28 miScript miRNA PCR Array Handbook 05 2012 Table 5 cDNA dilution prior to PCR PCR application Array Reaction dilution Pathway profiling Pathway Focused Add 200 ul RNase free water to miScript miRNA each 20 ul reverse transcription PCR Arrays reaction High content miScript miRNA HC Add 90 ul RNase free water to each pathway profiling PCR Arrays 20 ul reverse transcription reaction Whole miRNome miRNome miScript Dilution depends on the number of profiling miRNA PCR Arrays plates Rotor Discs in the miRNome miScript miRNA PCR Array For 1 x 384 well plate or 4 x 96 well plates Rotor Discs add 90 ul RNase free water to the 20 ul reverse transcription reaction For 2
64. ssue miRNA For 50 RNA preps PAXgene RNA 766134 Kit 50 MinElute Spin Columns PAXgene Shredder Spin Columns Processing Tubes Microcentrifuge Tubes Carrier RNA RNase Free DNase and RNase Free Buffers to be used with PAXgene Tissue Containers PAXgene Tissue For collection fixation and stabilization 765112 Containers 10 of 10 samples 10 Prefilled Reagent Containers containing PAXgene Tissue Fix and PAXgene Tissue Stabilizer PAXgene Blood miRNA For 50 RNA preps PAXgene Spin 763134 Kit 50 Columns PAXgene Shredder Spin Columns Processing Tubes Microcentrifuge Tubes RNase Free DNase RNase Free Reagents and Buffers to be used with PAXgene Blood RNA Tubes available from BD cat no 762165 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor 58 miScript miRNA PCR Array Handbook 05 2012 Trademarks QIAGEN QIAzol QIAgility HotStarTaq miScript QuantiTect RNeasy Rotor Gene Rotor Disc MinElute QIAGEN Group PAXgene PreAnalytiX GmbH Roche LightCycler Roche Group ABI PRISM Applied Biosystems StepOnePlus ROX SYBR Life Technologies Corporation Eppendorf Mastercycler Eppendorf AG Stratagene Mx3005P Mx3000P Mx4000 Agil
65. ter Mix miScript Universal Primer 10x miScript Primer Assay contains one miRNA specific primer Array of assays for a pathway disease or gene family for human mouse rat dog or rhesus macaque miRNAs available in 96 well 384 well or Rotor Disc 100 format High content array of assays for a pathway disease or gene family miRNAs available in 384 well format Array of assays for the complete human mouse rat dog or rhesus macaque miRNome available in 96 well 384 well or Rotor Disc 100 format Visit www giagen com GeneGlobe to search for and order these products 56 Cat no 218160 218161 218073 218075 Varies Varies Varies miScript miRNA PCR Array Handbook 05 2012 Product miScript miRNA QC PCR Array RT PCR Array Loading Reservoir 384EZLoad Covers Related products miScript PreAMP PCR Kit 12 miScript PreAMP PCR Kit 60 miScript PreAMP Primer Mix miRNeasy Micro Kit 50 miRNeasy Mini Kit 50 miRNeasy 96 Kit 4 Contents Array of quality control assays for human mouse rat dog or rhesus macaque miRNAs available in 96 well 384 well or Rotor Disc 100 format 12 x 5 ml capacity irradiation sterilized loading reservoirs Pack of 4 color coded covers for loading 384 well plates For 12 preamplification reactions 5x miScript PreAMP Buffer HotStarTaq DNA Polymerase 2 U ul miScript PreAMP Universal Primer 4 miScript Primer Assa
66. ting amount a single reverse transcription reaction can provide sufficient cDNA for 8 x 384 well plates or 32 x 96 well plates Rotor Discs Procedure 1 Thaw template RNA on ice Thaw RNase free water 10x miScript Nucleics Mix and 5x miScript HiSpec Buffer at room temperature 15 25 Mix each solution by flicking the tubes Centrifuge briefly to collect residual liquid from the sides of the tubes and then store on ice 2 Prepare the reverse transcription reaction on ice according to Table 4 miScript miRNA PCR Array Handbook 05 2012 27 Gently mix and then store on ice The reverse transcription master mix contains all components required for first strand cDNA synthesis except template RNA Note miScript Reverse Transcriptase Mix should be removed from the 20 freezer just before preparation of the master mix gently mixed and placed on ice It should be returned to the freezer immediately after use Note If setting up more than one reaction prepare a volume of master mix 1096 greater than that required for the total number of reactions to be performed Table 4 Reverse transcription reaction components Component Volume reaction 5x miScript HiSpec Buffer 4 ul 10x miScript Nucleics Mix 2 ul RNase free water Variable miScript Reverse Transcriptase Mix 2 ul Template RNA added in step 3 Variable see Table 3 for recommendations Total volume 20 pl If RNA was prepared from 100 200 ul serum or pla
67. tion has been diluted appropriately See Table 5 for recommendations If cDNA has been preamplified using the miScript PreAMP PCR Kit with a miScript PreAMP Primer Mix see the miScript PreAMP Handbook for dilution recommendations Do not vortex template cDNA or any of the components of the miScript SYBR Green PCR Kit The standard miScript miRNA PCR Array reaction volumes are 10 ul per well for a 384 well plate 25 ul per well for a 96 well plate and 20 ul per well for a 100 well Rotor Disc The miScript SYBR Green PCR Kit 200 provides sufficient reagents for 4 x 96 well arrays 110 residual 2 x 384 well pathway arrays 1210 ul residual 2 x 384 well HC miRNome arrays 1510 ul residual or 5 Rotor Disc 100 arrays 110 residual The miScript SYBR Green PCR Kit 1000 provides sufficient reagents for 20 x 96 well arrays 0 ul residual 12 x 384 well pathway arrays 1100 residual 13 x 384 well HC miRNome arrays 850 ul residual or 25 Rotor Disc 100 arrays ul residual M If using the iCycler iQ iQ5 or MyiQ well factors must be collected at the beginning of each experiment Well factors are used to compensate for any system or pipetting nonuniformity For details refer to the user manual supplied with the instrument or Technical Information Using QuantiTect SYBR Green Kits on Bio Rad cyclers available at www giagen com 30 miScript miRNA PCR Array Handbook 05 2012 The miScript miRNA QC PCR Array
68. trols miScript PCR control Primer Assay Primer Assay Figure 8 miScript miRNA QC PCR Array layout for plate formats E and Wells 1 to 4 of each row contain replicate C elegans miR 39 miScript Primer Assays 39 Wells 5 and 6 of each row contain replicate miR 16 miScript Primer Assays 16 Wells 7 and 8 of each row contain replicate miR 21 miScript Primer Assays 21 Wells 9 and 10 of each row contain replicate miR 191 miScript Primer Assays 191 Wells 11 to 16 of each row contain assays for different snoRNAs 61 SNORD61 assay 95 SNORD 95 assay 96 SNORD96A assay Wells 17 to 20 of each row contain replicate miRTC miScript Primer Assays miRTC Wells 21 to 24 of each row contain replicate positive PCR controls PPC These formats enable the quality assessment of up to 32 cDNA samples 20 miScript miRNA PCR Array Handbook 05 2012 Well 1 empty II A 90 miRTC eO snoRNA miRNA y C elegans 6 Postive PCR control 7 s miRTC miScript Primer Assay snoRNA miScript PCR Controls m E miScript Primer Assays e C elegans miScript Primer Assay e E e A 5 6 e a Peg 3 009 Figure 9 miScript miRNA QC PCR Array layout for Rotor Disc format Wells 1 and 2 contain replicate C elegans miR 39 miScript Primer Assays Well contains a miR 16 miScript Primer Assay Well 4 contains a miR 21 miScript Primer Assay Well 5 contains miR 191 miScript Primer Assay
69. ts are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated The purchaser and user of the kit agree to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and or its components For updated license terms see www giagen com 2011 2012 QIAGEN all rights reserved miScript miRNA PCR Array Handbook 05 2012 59 MIM I www qiagen com Australia techservice au qiagen com Austria techservice at qiagen com Belgium techservice bnI qiagen com Brazil suportetecnico brasil qiagen com Canada techservice ca Qqiagen com China techservice cn Qqiagen com Denmark techservice nordic Qqiagen com Finland techservice nordic qiagen com France techservice fr qiagen com Germany techservice de qiagen com Hong Kong techservice hk qiagen com India techservice india Qqiagen com Ireland techservice uk Qqiagen com Italy techservice it qiagen com Japan techservice jp qiagen com Korea South techservice kr qiagen com Luxembourg techservice bnI qiagen com Mexico
70. ys RNase Free Water For 60 preamplification reactions 5x miScript PreAMP Buffer HotStarTaq DNA Polymerase 2 U ul miScript PreAMP Universal Primer 4 miScript Primer Assays RNase Free Water Primer mix for preamplification for use with corresponding miScript miRNA PCR Array For 50 total RNA preps 50 RNeasy MinElute Spin Columns Collection Tubes 1 5 ml and 2 ml QIAzol Lysis Reagent RNase Free Reagents and Buffers For 50 preps 50 RNeasy Mini Spin Columns Collection Tubes 1 5 ml and 2 ml QIAzol Lysis Reagent RNase Free Reagents and Buffers For 4 x 96 preps 4 RNeasy 96 plates Collection Microtubes racked Elution Microtubes CL Caps S Blocks AirPore Tape Sheets QIAzol Lysis Reagent RNase Free Reagents and Buffers miScript miRNA PCR Array Handbook 05 2012 Cat no Varies 338162 338125 331451 331452 Varies 217084 217004 217061 57 Product Contents Cat no miRNeasy For 50 total RNA preps 50 RNeasy 217184 Serum Plasma Kit 50 MinElute Spin Columns Collection Tubes 1 5 ml and 2 ml GlAzol Lysis Reagent Ce miR 39 1 miScript Primer Assay RNase free Reagents and Buffers miRNeasy 10 pmol lyophilized C elegans miR 39 219610 Serum Plasma Spike In miRNA mimic Control miRNeasy FFPE Kit 50 50 RNeasy MinElute Spin Columns 217504 Collection Tubes Proteinase K RNase Free DNase DNase Booster Buffer RNase Free Buffers RNase Free Water PAXgene Ti
71. ys for miR 16 miR 21 and miR 191 are examined Note Mature miRNAs miR 16 miR 21 and miR 191 are ubiquitously expressed in a wide range of cells tissues and body fluid samples including whole blood serum and plasma 9 C values of the miScript PCR Controls for SNORD61 SNORD95 and SNORD96A are examined Note These small RNAs have been verified to have relatively stable expression levels across tissues and cell types Nevertheless snoRNA snRNA control values remain sample dependent and should be checked to determine whether their expression is consistent across the samples that are being analyzed In particular snoRNAs and long noncoding RNAs are usually not present in serum plasma urine and other body fluids If the expression of a particular control is not consistent across experimental 44 miScript miRNA PCR Array Handbook 05 2012 samples that control should not be used for data normalization For examples of C values associated with various tissue types refer to the miScript PCR Controls application data at www giagen com miRNAControls 10 C elegans miR 39 miScript Primer Assay values Ce are examined The C elegans miR 39 miScript Primer Assay should only result in values above the threshold if the miRNeasy Serum Plasma Spike In Control Syn cel miR 39 miScript miRNA Mimic has been spiked in to the sample prior to RNA purification For more information see the miRNeasy Serum Plasma Handbook 1

Sample & Assay Technologies miScript® miRNA PCR

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Sample &amp; Assay Technologies miScript&reg; miRNA PCR

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Sample & Assay Technologies miScript® miRNA PCR