miProfile miRNA qPCR array User Manual
Contents
1. og Adct The value of 24 is the change in expression level of the gene of interest between different samples Vil Appendix Il Note for most updated catalog array list please visit http www genecopoeia com product mirna solutions mirna qpcr arrays Cat No qPCR array products Quantity set Shipping and storage condition 1 565 miRNAs Shipped at room temperate PAM HG96 mews DOr eaten 19 x 96 well plates Stable for at least 6 months when miRNA qPCR arrays o stored at 20 C F i F Shipped at room temperate miProfile human single nucleotide 61 miRNAs FANDEISSD mismatch miRNA qPCR arrays 1 x 96 well plate Rear brad monthswhen Shipped at room temperate miProfile human cancer miRNA 420 miRNAs PAM HC96 qPCR arrays 5 x 96 well plates n Pad 6 months when Shipped at room temperate PAMEHCNGS i rene human brain cancer 84 miRNAs Stable for at least 6 months when miRNA qPCR arrays 1 x 96 well plate stored at 20 C Shipped at room temperate miProfile human breast cancer 168 miRNAs PAM HCB96 Stable for at least 6 months when meat miRNA qPCR arrays 2 x 96 well plate stored at 20 C miProfile human leukemia miRNA 168 miRNAs Shipped at room temperate PAM HCX96 Stable for at least 6 months when qPCR arrays 2 x 96 well plate stored at 20 C Shipped at room temperate Stable for at least 6 months when stored at 20 C miProfile human lung cancer 168 miRNAs PAM HCL96 miRNA qPCR arrays 2 x 962. been pre validated and deposited in designated wells Each plate also has 12 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 qPCR 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 hi
3. miRNAs 2 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 168 miRNAs 2 x 96 well plate 11 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate Stable for at least 6 months
4. 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 other 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 refun
5. when stored at 20 C Shipped at room temperate Stable for at least 6 months when PAM MG96 PAM MX96 miProfile miRNA PCR Array User Manual miProfile mouse miRNome miRNA qPCR arrays miProfile mouse breast cancer miRNA qPCR arrays miProfile mouse brain cancer miRNA qPCR arrays miProfile mouse ovarian cancer miRNA qPCR arrays miProfile mouse prostate cancer miRNA qPCR arrays miProfile mouse colorectal cancer miRNA qPCR arrays miProfile mouse carcinoma miRNA qPCR arrays miProfile mouse lung cancer miRNA qPCR arrays miProfile mouse melanoma miRNA qPCR arrays miProfile mouse pancreatic cancer miRNA qPCR arrays miProfile mouse head and neck cancer miRNA qPCR arrays miProfile mouse leukemia miRNA qPCR arrays miProfile mouse inflammatory miRNA qPCR arrays miProfile mouse heart disease miRNA qPCR arrays miProfile mouse immunopathology miRNA qPCR arrays miProfile mouse serum and plasma miRNA qPCR arrays 834 miRNAs 10 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well p
6. NAzol amp RT RNA isolation Eo mi Stable for at least two years when stored at reagent room temperature Shipped with dry ice AMRT 0020 All in One miRNA first 20 reactions te Store at 20 C Stable for at least 12 AMRT 0060 strand cDNA synthesis kit 60 reactions months Alternatively store at 80 C in aliquots Avoid repeated freezing thawing AOPR 0200 200 reactions Shipped with dry ice AOPR 1000 All in One qPCR mix 1000 reactions Store at 20 C Stable for at least 12 AOPR 4000 4000reactions months Alternatively store at 80 C in aliquots Avoid repeated freezing thawing Estimates of number of RT PCR reactions required for EACH SAMPLE Numbers of plates per Numbers of RT reactions Numbers of PCR reactions sample per sample per sample pe Array format 96 well plate 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 6 miProfile miRNA PCR Array User Manual 10 ul to 1 000 ul adjustable single channel micropipettes with disposable tips 5 ul to 20 yl adjustable multichannel micropipette disposable tips and reservoir qPCR instrument compatible with miRNA qPCR arrays ordered Preparation Important notes 1 2 Before use remove any condensation that has accumulated on the plate sealing surface and c
7. cate plates were plotted against each other R2 gt 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 5 les 80 o 2 o 60 e V 40 E p 20 ac 1 95 0 miR 29a miR 29c B Target Specific Assay m Off Target Assay A 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 Product Array Layout and Array Format Options Catalog miProfile miRNA qPCR arrays See the complete list in Appendix II or visit http www genecopoeia com product mirna solutions mirna qpcr arrays Array format options GeneCopoeia provides five qPCR array formats A B C D and E suitable for use with the following real time cyclers Important note Upon receiving please check to make sure that the correct array format was ordered to ensure the compatibility with your qPCR instrument Plate format Instrument provider qPCR instrument model A 5700 7000 7300 7500 7700 7900HT Standard 96 well 96 we
8. d 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 Inc NanoDrop Thermo Scientific PAM062813 13
9. entrifuge plates briefly to collect the contents to the bottom of the plate wells Strictly follow the standard procedures for PCR to avoid nucleic acid contamination and non specific amplifications 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 Array format plates per recommended recommended reactions per reactions per sample per sample per sample sample sample 1 5 3 ug 5 10 ug 3 2 s m stove mue h foz fosos RNA quantification and quality control 1 2 3 IV 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 Use the formula A260 x dilution x 40 ug RNA mL to determine the RNA concentration Check the RNA integrity by agarose electrophoresis 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 2 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 Prepare miRNA polyA polymerase PAP and reverse transcriptase RT reaction mix Add t
10. es and then place them on ice Remove any condensation that has accumulated 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 2 Prepare qPCR solution on ice Components 1 well N well 2xAll in One qPCR Mix 10ul 11pl x N miRNA cDNA 10 times dilution ipl 1 1ul x N 50 X Rox Reference Dye 0 4ul 0 44ylx 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 D 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 3 Mix the qPCR solution thoroughly and centrifuge briefly Accurately transfer exactly 20 yl reaction mix to each well Change tips after each transfer to avoid cross contamination 4 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 5 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 Exten
11. gh throughput 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 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 N gt As Sample miRNA cDNA control miRNA cDNA B Add qPCR Mix and cDNA to the qPCR Array Plate id ed edd eee oe p HTHEHE rod Change Teast Homo Brain Assay Data Sample Homo Uver Assay Data Control Stee Larue eneee tetas prae ete cwn eiae A Total Small RNA amp Mature miRNA Rods 3 Polyadenylation AAAAAAAAAA nnn 3 gt cDNA r
12. he following reagents to the ice chilled RNase free reaction tubes to a final total volume of 25 ul Component Volume Quantity Small RNA 0 5 1 0 yg 2 5U ul PolyA Polymerase 1 ul 2 5U RTase mix 1 ul 5xPAP RT buffer 5 ul 1x Spike in RT control 1 ul ddH2O 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 b To increase the rate of positive detection an input of 0 5 1 0ug of small RNA is recommended for miProfile miRNA PCR Array User Manual the standard miRNA reverse transcription reaction 25 ul 3 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 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 tub
13. late 12 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 Shipped at room temperate 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 miProfile miRNA PCR Array User Manual Vill Limited Use License and Warranty Limited use license Following terms and conditions apply to use of miProfile miRNA
14. ll Applied Biosystems block ViiA 7 Standard 96 well block B 7500 Fast block 7900HT Fast block StepOnePlus 96 well Applied Biosystems ViiA 7 Fast block ae Bio Rad Laboratories iCycler iQ MyiQ iQ 5 D i CFX96 DNA Engine Opticon DNA Engine Opticon 27 96 well Bio Rad Laboratories Ghromo4 cud Roche Applied Science LightCycler 480 96 well block miProfile miRNA PCR Array User Manual Array layout Figure 5 Illustration of miProfile miRNA qPCR array layout 96 well plate e miRNA primer pairs Wells 1 84 are designated 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 RNA extraction and RT PCR reagents required sold separately Cat No Products Quantity set Shipping and storage condition Shipped at room temperature E01010A R
15. m M Expressway fo po very miProfile miRNA PCR Arrays 96 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 lll Preparation IV Procedure V Data Analysis VI Appendix VII Appendix II VIII Limited Use License and Warranty I 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 96 well plate contains up to 84 pairs of PCR primers forward miRNA specific primer reverse universal primer which have
16. o M TTTTTTTTT cDNA template ready for qPCR All in One miRNA qPCR primer o 5 _ a e B eo Univesal Adaptor PCR Primer Reverse transcription 2___ A 2 qPCR Assay Figure 1 miRNA PCR array experiment work flow A and miRNA RT PCR mechanism B miProfile miRNA PCR Array User Manual Performance data 35 an 23 5 C value 15 10 20 Linear Range and Sensitivity Total RNA m miR 21 miR 1260 y 3 3054x 25 379 R 0 9969 y 3 118x 24 479 R 0 9985 1 0 1 2 3 4 Logo 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 40 35 30 C value of plate B N n Inter Array Reproducibility y 0 9864x 0 1491 R 0 9985 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 repli
17. om cycle 2 to two cycles before the earliest visible amplification Normally 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 Setthreshold 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 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 Qc 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
18. sion 72 C 10 sec Yes a The DNA polymerase used in the 2X All in One qPCR Mix is a special chemically modified hot start 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 qPCR cycling Temperature range Heating rate Constant temperature Detection 66 C 95 C 0 5 C unit time 6sec unit time Yes miProfile miRNA PCR Array User Manual 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 fr
19. the thresholds have been correctly defined the value of Ct of PCR should be 2032 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 2033 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 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 anal
20. well plate 10 miProfile human serum and PAMA plasma miRNA qPCR arrays miProfile miRNA PCR Array User Manual miProfile human ovarian cancer miRNA qPCR arrays miProfile human bladder cancer miRNA qPCR arrays miProfile human colorectal cancer miRNA qPCR arrays miProfile human endometrial cancer miRNA qPCR arrays miProfile human gastric cancer miRNA qPCR arrays miProfile human hepatocellular carcinoma miRNA qPCR arrays miProfile human lymphoma miRNA qPCR arrays miProfile human melanoma miRNA qPCR arrays miProfile human head and neck cancer miRNA qPCR arrays miProfile human pancreatic cancer miRNA qPCR arrays miProfile human prostate cancer miRNA qPCR arrays miProfile human inflammatory miRNA qPCR arrays miProfile human heart disease miRNA qPCR arrays miProfile human immunopathology miRNA qPCR arrays miProfile human IPS stem cell miRNA qPCR arrays miProfile human muscle disease miRNA qPCR arrays miProfile human toxicology related miRNA qPCR arrays 168 miRNAs 2 x 96 well plate 79 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 80 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 168 miRNAs 2 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 84 miRNAs 1 x 96 well plate 168
21. ysis results allow genes of interest to be simply and rapidly selected for further study VI Appendix I miProfile miRNA PCR Array User Manual 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 Ct 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 x2 SX N x 2 CH 2 Ctr g 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 Nrela 2 C12 AC 2 Actt ACt2
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