miProfile™ miRNA qPCR arrays user manual (384
Contents
1. 0 99 were observed for high inter array reproducibility R2 gt 0 99 is also observed for intra array reproducibility data not shown miProfile miRNA PCR Array User Manual Single Nucleotide Mismatches 120 100 00 100 00 100 5 80 O g 60 a V 40 E i 20 v oc 1 95 0 miR 29a miR 29c O Target Specific Assay m Off Target Assay A rain oaceae B Figure 4 Specificity of miRNA detection miRNA miR 29a and miR 29c with one single nucleotide mismatch B can be distinguished Relative detection defined as a percentage of the perfect match 100 x 2 ACt was calculated using the Ct values of on target and off target assays which were performed to detect miRNA plasmid DNA templates using All in One miRNA qRT PCR Detection Kits A ll Products Array Layout and Array Format Options Shipping and storage condition Cat No qPCR array products Quantity set 1 700 miRNAs Shipped at room temperate 5 x 384 well plates Stable for at least 6 months when stored at 20 C Shipped at room temperate Stable for at least 6 months when stored at 20 C PAM HG384 miProfile human miRNome miRNA qPCR arrays miProfile human single nucleotide 834 miRNAs FAM MGS4 mismatch miRNA qPCR arrays 3 x 384 well plate RNA extraction RT PCR reagents required sold separately Cat No Products Quantity set Shipping and storage condition RNAzol RT RNA Shipped at RT E01010A Isolation2. Inc NanoDrop Thermo Scientific PAM062813 11
3. M Expressway to po very miProfile miRNA PCR Arrays 384 Well For high throughput profiling of miRNA expression User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 support genecopoeia com www genecopoeia com 2013 GeneCopoeia Inc miProfile miRNA PCR Array User Manual USER MANUAL miProfile miRNA PCR Array I Introduction Il Kit Components and Array Format Options Ill Preparation IV Procedure V Data Analysis VI Appendix VII Limited Use License and Warranty l Introduction MicroRNAs miRNAs are small non coding RNAs that regulate gene expression at the post transcriptional level Usually 21 23 nucleotides in length miRNAs are important modulators in cellular pathways and are highly conserved in eukaryotic organisms Irregularities in miRNA regulated gene expression have been found to be associated with cancers cardiovascular disorders and a variety of other diseases The miProfile miRNA PCR Arrays are designed for profiling the expressions of pre defined or customized sets of miRNAs in various tissues or cells The resulting differential expressions of profiled miRNAs help researchers to identify those miRNAs that are of biological significance and importance relevant to their research Each 384 well plate contains up to 360 pairs of PCR primers forward miRNA specific primer reverse universal primer which have been pre vali
4. Reagent Stable for at least 2 years when stored at RT AMRT 0020 All in One miRNA first 20 reactions AMRT 0060 strand cDNA synthesis kit 60 reactions Shipped with an ice pack Stable for at least 6 months when stored at 20 C AOPR 0200 200 reactions Shipped with an ice pack AOPR 1000 All in One qPCR mix 1000 reactions Stable for at least 6 months when stored at AOPR 4000 4000reactions 20 C miProfile miRNA PCR Array User Manual Array format options Important note Upon receiving please check to make sure that the correct array format was ordered to ensure the compatibility with your qPCR instrument GeneCopoeia provides five qPCR array formats A B C D and E suitable for use with the following real time cyclers Plate format Instrument provider qPCR instrument model a 4 well Applied Biosystems 7900HT 384 well block ViiA 7 384 well block Caen Bio Rad Laboratories CFX384 384 well block Cee aoe LightCycler 480 384 well block Array layout is u5 16 17 18 20 21 22 23 24 EE PN EEE 25 26 27 28 20 30 31 32 33 34 35 36 37 38 39 40 a1 42 43 aa a5 46 7 48 49 50 51 52 53 54 s5 56 57 se 50 60 61 62 63 64 es 66 67 68 69 70 71 72 73 74 75 76 77 78 79 so 81 82 83 84 e5 96 87 88 89 90 91 92 93 94 o5 96 97 98 99 100 10
5. 1 102 103 104 105 106 107 108 109 140 144 112 113 144 115 116 117 118 119 120 121 122 123 126 125 126 127 128 120 130 131 132 133 134 196 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 154 162 153 154 165 156 157 158 159 160 161 162 163 184 165 166 167 168 169 170 174 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 214 212 213 214 215 216 217218 219 220 221 222 223 224 225 228 227 228 220 230 231 232 233 234 236 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 23 254 256 256 257 268 259 260 261 262 263 264 266 266 267 268 289 270 274 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 201 292 293 294 206 296 297 298 209 300 301 302 303 304 305 306 307 308 309 310 311 3121 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 320 330 331 332 333 334 395 336 BM nc No nc nc kt ict Ke Ka HK HK HK kak HK HK HKG RT RT RT RT PCR PCR PCR PCR Figure 5 Illustration of miProfile miRNA qPCR array layout 384 well plate i A N J Er a 4 e miRNA primer pairs Wells 1 360 are designate
6. ally it is between 2 to 10 cycles Do not use cycles greater than 15 Ensure that baseline settings are the same across all PCR runs in the same analysis to allow comparison of results 2 Set threshold Correct placement of the threshold is the next crucial step in data analysis To adjust the threshold properly set the threshold value within the exponential phase of all amplification plots when viewed using the logarithmic scale for the y axis Generally the expression level of each reference gene should be higher than most other genes 3 Obtain the Ct or Cp values The Ct is defined as the cycle when sample fluorescence exceeds a chosen threshold above background fluorescence This is also known as the Cp or crossing point 4 Export the data Most qPCR instruments provide a function for exporting Ct or Cp values to Excel 5 Analyze the qPCR results using the AACT method of relative quantification and interpretation of the control wells 6 All Ct values reported as greater than 35 or as N A not detected are considered as not detectable 1 Examined amplification and melting status of each gene using the qPCR instrument software Each reference gene RTC and PPC should exhibit only one melting peak per reaction 2 Examined CT values of the positive PCR control wells PCR 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 Ct of PCR shoul
7. d be 20 2 across all arrays or samples 3 Examined CT values of the positive RT control wells RT 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 Ct of RT should be 203 across all arrays or samples Data analysis Analyze the qPCR result with GeneCopoeia s online Data Analysis System free which is available at http www genecopoeia com product gpcr analyse miProfile miRNA PCR Array User Manual This Data Analysis System uses the AAC method to perform fold change analysis or simple statistical analysis of the expression level C or Cp values for each gene 1 Download and read the Primer Array Date Analysis Operation Guide before performing analysis 2 Import the C or Cp values into the corresponding data analysis template form Sample Data xls and Control Data xls Upload the template form and choose the correct reference and analysis factors Note The reference factor chosen for qPCR Primer Array for normalization with the AAC method must not be influenced by the experimental design Therefore use one or more factors that have been previously verified experimentally A single value or an average of the C values for the reference factor can be used for normalization 3 Perform the specified analysis When a test is repeated at least three times statistical results p value are provided The analysis results allow genes o
8. d wells for pre deposited miRNA primer pairs NC Negative controls which only have the pre deposited reverse universal primers e HK1 6 Six pre deposited housekeeping snRNAs HK1 6 primer pairs which can be used as endogenous positive controls as well as for array normalization e RT Spike in reverse transcription controls which can be used to monitor the efficiency of the RT reactions These pre deposited primer pairs specifically amplify the cDNA template reversed transcribed from the spike in exogenous RNA in the sample e PCR Positive PCR controls which are used to verify the PCR efficiency by amplifying the pre deposited DNA template with its specific pre deposited primer pairs Other materials required but not provided Small total RNA extraction kit i e RNAzol RT DNase RNase free tips PCR reaction tubes 1 5 ml microcentrifuge tubes 5 ml and 10 ml graduated pipettes beakers flasks and cylinders 10 ul to 1 000 ul adjustable single channel micropipettes with disposable tips 5 ul to 20 ul adjustable multichannel micropipette disposable tips and reservoir qPCR instrument compatible with miRNA qPCR arrays ordered miProfile miRNA PCR Array User Manual Estimates of number of RT PCR reactions required for EACH SAMPLE Numbers of PCR reactions Array format Numbers of plates Numbers of RT reactions lll Preparation Important notes 1 Before use remove any condensation that has accumulated on the plate seal
9. dated and deposited in designated wells Each plate also has 24 wells that contain different types of controls for monitoring the efficiency of the entire experimental process from reverse transcription to qPCR reaction The All in One miRNA First Strand cDNA Synthesis Kits AMRT 0020 AMRT 0060 and qPCR Mix Kits AOPR 0200 AOPR 1000 AOPR 4000 are the recommended RT PCR reagents for use with the miProfile miRNA gPCR arrays These reagents have been optimized to produce high sensitivity efficiency and specificity The All in One reverse transcriptase mix contains a novel and optimized blend of polyA polymerase and reverse transcriptase in a buffer that allows high activities and maximal performances of both enzymes In such reactions the polyA polymerase adds poly A tails to mature miRNAs to generate polyA miRNAs In the same reaction m MLV RTase and a unique oligo dT adaptor primer compatible with the PCR universal reverse primer pre deposited in the miRNA plates reverse transcribe the polyA miRNAs The All in One qPCR Mix containing SYBR Green is used to specifically detect the reverse transcribed miRNA with the miRNA specific forward primer and PCR universal reverse primer which are pre deposited in the miRNA plates Similar reagents from third party vendors may be compatible for use However their uses are not supported Using a universal real time PCR condition one can easily profile and analyze the miRNA expression in a high throughp
10. er than the original purchaser of the Product Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose GeneCopoeia is committed to providing our customers with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2013 GeneCopoeia Inc GeneCopoeia Inc 9620 Medical Center Drive Suite 101 Rockville MD 20850 1 301 762 0888 1 866 360 9531 support genecopoeia com GeneCopoeia Products are for Research Use Only Copyright 2013 GeneCopoeia Inc Trademarks GeneCopoeia miProfile All in One miProfile GeneCopoeia Inc SYBR Molecular Probes iCycler iQ MyiQ iQ 5 CFX96 DNA Engine Opticon DNA Engine Opticon 2 Chromo4 Bio Rad LightCycler Roche Trizol ABI ROX ViiA StepOnePlus Life Technologies RNAzol Molecular Research Center
11. evel of the gene of interest between different samples 10 miProfile miRNA PCR Array User Manual Vil Limited Use License and Warranty Limited use license Following terms and conditions apply to use of miProfile miRNA qPCR Arrays the Products If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not be resold repackaged or modified for resale or used to manufacture commercial products or deliver information obtained in service without prior written consent from GeneCopoeia This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited warranty GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia will provide the purchaser with a refund This limited warranty shall not extend to anyone oth
12. f interest to be simply and rapidly selected for further study VI Appendix AACt data analysis method AAC data analysis a relative quantitative analysis technique is the most simple and direct method for gene expression analyses The method requires stable expression from a reference gene to normalize the variation introduced by each step including sample collection RNA isolation reverse transcription and amplification Typically housekeeping genes are used as reference genes In qPCR as in any amplification based technique the number of amplification products N is calculated as follows N NOx 1 E NO number of template molecules Cr threshold cycle E amplification efficiency When the amplification efficiency E is 100 the number of template molecules in pre amplification mix is calculated as follows NO Nx2 To analyze the change in expression level for the gene of interest in multiple samples using the AAC method the amount of the amplification template from different samples is normalized by dividing the expression level of the gene of interest x with the reference factor r as follows Nrel NOx NOr N x 2 SX N x 2 OH atm Ct _ 5 Act The change in normalized expression levels of the gene of interest x between experimental sample sample 1 and the control sample sample 2 is as follows Nrel Nreloa 24 1 2 4C2 9 Actt Act2 E gAACt AACt The value of 2 is the change in expression l
13. inal total volume of 25 ul 3 miProfile miRNA PCR Array User Manual Component Volume Quantity Small RNA 0 5 1 0 pg 2 5U ul PolyA Polymerase 1ul 2 5U RTase mix 1ul 5xPAP RT buffer 5 ul 1x Spike in RT control Tul ddH20 RNase DNase free to final 25ul a cDNA product from a standard miRNA reverse transcription reaction 25 ul should be enough for 2 plates of 96 Well reactions Prepare at least 10 standard miRNA reverse transcription reactions for the 19 plates of whole genome miRNA PCR Arrays To increase the rate of positive detection an input of 0 5 1 0ug of small RNA is recommended for the standard miRNA reverse transcription reaction 25 ul Perform reverse transcription reaction Mix the prepared reaction mix gently by pipetting up and down Incubate at 37 C for 60 minutes Terminate the reaction by incubating at 85 C for 5 minutes After the incubation dilute the cDNA products 10 times by adding 225ul of sterile water to each RT reaction and use it for the subsequent qPCR reactions The diluted cDNA can be stored at 20 C for several weeks o qPCR reaction Note Be sure the miProfile miRNA PCR Array plate is compatible with your qPCR instrument before beginning this protocol 1 Thaw the reagents of All in One miRNA qPCR Mix Kit Invert the tubes to mix gently but thoroughly Briefly centrifuge to bring the contents to the bottom of the tubes and then place them on ice Remove any condensation that has accu
14. ing surface and centrifuge plates briefly to collect the contents to the bottom of the plate wells 2 Strictly follow the standard procedures for PCR to avoid nucleic acid contamination and non specific amplifications 3 Read the instructions thoroughly before attempting to perform the procedures Estimates of RNA and number of RT PCR reactions required for EACH SAMPLE Number of Small RNA Total RNA Number of RT Number of qPCR plates recommended recommended reactions reactions Array format 384 well plate 5 10 ug 10 20 ug 2250 RNA quantification and quality control 1 Dilute the RNA sample with the RNase free water and measure the absorbance at 260 nm and 280 nm A260 280 should be greater than 1 8 2 Use the formula A260 x dilution x 40 ug RNA mL to determine the RNA concentration 3 Check the RNA integrity by agarose electrophoresis IV Procedure First strand cDNA synthesis Note High quality cDNA is a prerequisite for accurate detection of miRNA expression GeneCopoeia s All in One miRNA First Strand cDNA Synthesis Kit is required for small cDNA synthesis 1 Thaw the reagents in All in One miRNA First Strand cDNA Synthesis Kit mix by gently flicking the tube briefly centrifuge to bring the contents to the bottom of the tubes and then place them on ice 2 Prepare miRNA polyA polymerase PAP and reverse transcriptase RT reaction mix Add the following reagents to the ice chilled RNase free reaction tubes to a f
15. mplate ready for qPCR B cDNA All in One miRNA qi VEE et Univesal Adaptor PCR Primer R primer qPCR Assay gt Figure 1 miRNA PCR array experiment work flow A and miRNA RT PCR mechanism B miProfile miRNA PCR Array User Manual Performance data Linear Range and Sensitivity Total RNA e o m miR 21 miR 1260 30 y 3 3054x 25 379 25 R 0 9969 v 3 S 20 Oo 15 y 3 118x 24 479 10 R 0 9985 5 Bi T T T T 2 1 0 1 2 3 4 Log ng of total RNA Figure 2 Broad linear range and high sensitivity Starting with serially diluted amounts of human colon cancer total RNA miR 21 and miR 1260 were detected using All in One miRNA qRT PCR Detection Kit The resulting Ct values were plotted against the log10 of the amounts of input total RNA The data demonstrated a broad linear dynamic range from 20pg to 2 ug of input total RNA as well as high sensitivity This allows the detection of miRNAs at varying expression levels including low expressers Inter Array Reproducibility 40 Ww wn Ww C value of plate B N N w y 0 9864x 0 1491 R 0 9985 w l 10 15 20 25 30 35 40 C value of plate A Figure 3 High inter array reproducibility Two miProfile PCR array replicates plate A and B were analyzed using human total RNA 10 tissue mix on the Bio Rad iQ5 The Ct values of the replicate plates were plotted against each other R2 gt
16. mulated on the plate sealing surface and centrifuge briefly to collect the contents to the bottom of the plate wells Carefully remove sealing film before use 96 Well qPCR Prepare qPCR solution on ice Components 1 well N well 2xAll in One qPCR Mix 10ul 11yl x N miRNA cDNA 10 times dilution 1ul 1 1pl x N 50 X Rox Reference Dye 0 4ul 0 44ulx N ddH20 m Not using Rox Reference Dye 9 ul 9 9 ulx N m Using Rox Reference Dye 8 6 ul 9 5 ulx N Final Volume 20ul 22ulx N a miProfile miRNA PCR Array is used to detect multiple miRNAs simultaneously in the same sample Ensure sufficient mix is available by preparing enough for the number of reactions to be used with a 10 additional volume for pipetting loss b 50xRox Reference Dye is added only for qPCR instruments that require ROX for calibration Mix the qPCR solution thoroughly and centrifuge briefly Accurately transfer exactly 20 ul reaction mix to each well Change tips after each transfer to avoid cross contamination Tightly seal the qPCR reaction plate with a new sealing film Ensure that the film seals smoothly to prevent refraction of light Centrifuge briefly to remove bubbles Run qPCR The following three step PCR program is recommended for running qPCR Cycles Steps Temperature Duration Detection 1 Initial denaturation 95 C 10min No 40 Denaturation 95 C 10sec No Annealing 60 C 20 sec No Extension 72 C 10 sec Yes a The DNA polymerase used in the 2X All in One
17. qPCR Mix is a special chemically modified hot start miProfile miRNA PCR Array User Manual enzyme The indicated initial denaturation is sufficient to activate the enzyme b The annealing temperatures of the cross linked primers in All in One qPCR Primer Array are designed and optimized For comparing the miRNAs with single nucleotide difference a higher annealing temperature 65 C might be necessary c The extension time indicated above is suitable for Bio Rad s iQ5 real time PCR instrument Adjust the time duration according to the documentation provided with your instrument When using SYBR Green dye to monitor the qPCR reaction a melting curve analysis should be performed immediately after GPCR cycling Temperature range Heating rate Constant temperature Detection 66 C 95 C 0 5 C unit time 6sec unit time Yes V Data Analysis 1 Define the baseline The baseline is the noise level in early cycles Each real time PCR instrument has algorithms to perform the baseline setting This may be a fixed number of cycles for all samples or adaptive for each sample depending on the type of instrument that is being used If the lowest Ct is less than the upper limit of the baseline setting then the baseline should be manually adjusted Use the Linear View of the amplification plot to determine the earliest visible amplification and then set the baseline from cycle 2 to two cycles before the earliest visible amplification Norm
18. ut fashion Small RNA is recommended as the input RNA to increase the specificity of detection although use of total RNA can achieve similar results Key advantages Genome wide coverage pre arranged groups or customized groups Largest genome wide miRNA coverage Cancer related groups Customized miRNA arrays for focused study Robust performance Sensitive Detect miRNAs from as little as 10 pg of input small RNA or 20 pg of total RNA Specific Capable of distinguishing miRNAs with single nucleotide mismatches Each primer set has been experimentally validated for specific amplification Broad linearity Allow miRNAs at different expression levels to be detected simultaneously Reproducible High reproducibility R gt 0 99 for inter array and intra array replicates 2 miProfile miRNA PCR Array User Manual Validated miRNA primers Each miRNA primer is designed using a proprietary algorithm and has been experimentally validated Protocol overview A Prepare cDNA from your RNA Samples ee control miRNA cDNA 9 Sample miRNA cDNA F B Add qPCR Mix and cDNA to the qPCR Array Plate C Perform real time PCR eet es D Analyze the qPCR Results with GeneCopoeia s Online Data Analysis System f asea 7 rees A e Total Small RNA amp 5 7 Mature miRNA Poly A polymerase E A AAAAAAAAA S 3 Polyadenylation PT 5 Reverse transcription a kel TTTTTTTTT CDNA te
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