ExProfile™ gene qPCR array user manual
Contents
1. ExProfile Gene qPCR Array User Manual Inter array reproducibility 40 00 y 0 9984x 0 1567 Des sand R 0 9919 Q amp Q 20 00 gt 4 gt 10 00 0 00 0 00 10 00 20 00 30 00 40 00 Ct value of plate A Figure 4 High inter array reproducibility Two ExProfile qPCR gene 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 R gt 0 99 was observed for high inter array reproducibility R gt 0 99 was also observed for intra array reproducibility data not shown ll Array Format and Layout 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 Apoled pigseiccac 5700 7000 7300 7500 7700 7900HT Standard 96 well 96 well PP y block ViiA 7 Standard 96 well block B Aed Bnawn 7500 Fast block 7900HT Fast block SteoOnePlus 96 well PP y ViiA 7 Fast block c T Tac iQT 96 well Bio Rad Laboratories iCycler iQ MyiQ iQ 5 D CFX96 DNA Engine Opticon DNA Engine Opticon 96 well Bio Rad Laboratories 2 Chromo4 ai 7900HT 382. Expressway to Discovery ExProfile Gene qPCR Arrays For high throughput profiling of coding gene 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 ExProfile Gene qPCR Array User Manual USER MANUAL ExProfile Gene qPCR Array Introduction ll Array Format and Layout Ill Arrays and Reagents IV Preparation V Procedure VI Data Analysis VII Appendix VIII Appendix II IX Limited Use License and Warranty I Introduction The ExProfile Gene qPCR Arrays are designed for profiling the expressions of pre defined or customized sets of coding genes in various tissues or cells The differential expressions of profiled genes help researchers to identify and or validate those that are biologically significant and important for their research For catalog arrays each 96 well plate contains up to 84 pairs of qPCR primers each 384 well plate contains up to 360 pairs of qPCR primers which have been pre validated and coated in designated wells In the same plate there are 12 wells in 96 well plate or 24 wells in 384 well plate that contain different types of controls for monitoring the efficiency of the entire experimental process from reverse transcription to GPCR reaction The All in One First Strand cDNA Synthesis Kits AORT 0020 AORT 0060 a
3. k ho JE fal BI BI TEM d MI j co fh Po F ch e RNA primer pairs Wells 1 84 are designated for a real time PCR assay for genes e HK1 6 Six pre deposited housekeeping gene HK1 6 primer pairs which can be used as endogenous positive controls as well as for array normalization e GDC Genomic DNA Controls which can be used to specifically detect genomic DNA contamination with a high level of sensitivity e RT Spike in RNA 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 lil Arrays and Reagents Catalog arrays For a complete list of catalog arrays please see Appendix II or visit following webpage http www genecopoeia com product qene qpcr array RT PCR and RNA extraction reagents sold separately ExProfile Gene qPCR Array User Manual Products Quantity set Shipping and storage condition Shipped with an ice pack Stable for at least 6 months when stored at 20 C AORT 0020 All in One first strand 20 reactions AORT 0060_ cDNA synthesis kit 60 reactions AOPR 0200 200 reactions Shipped with an ice pack AOPR 1000 All in
4. One qPCR mix 1000 reactions Stable for at least 6 months when stored at AOPR 4000 4000 reactions 20 C Shipped at room temperature Stable for at least two years when stored at room temperature RNAzol RT RNA EOTOI0A isolation reagent Other materials required but not provided Total RNA extraction kit RNAzol RT RNA extraction reagent is recommended 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 gene qPCR arrays ordered IV Preparation Important notes 1 Before use remove any condensation that has accumulated on the plate sealing surface and centrifuge plates briefly to collect the contents to the bottom of the plate wells 2 Strictly follow the standard procedures for qPCR 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 Total RNA Number of RT Number of qPCR recommended reactions per reactions per per sample sample sample Number of plates Array format per sample 96 well plate RNA quantification and quality control 384 well plate i s be 3
5. 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 IQP 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 PAG062613 15
6. wooo o wo o mo pew fo ooo bw gt 2o o oe 5 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 7 2 3 ExProfile Gene qPCR Array User Manual Use the formula A260 x dilution x 40 ug RNA mL to determine the RNA concentration Check the RNA integrity by agarose electrophoresis Genomic DNA contamination control The Genomic DNA Control GDC in each ExProfile gene qPCR array specifically tests for genomic DNA contamination in each sample during each qPCR performance A Ct value of genomic DNA control less than 35 indicates the presence of a detectable amount of genomic DNA contamination that should be addressed So it is necessary to remove genomic DNA and all residual contamination from your RNA samples V Procedure First strand cDNA synthesis Note High quality cDNA is a prerequisite for accurate detection of gene expression It is important to remove genomic DNA and all residual contamination from your RNA samples before using our All in One First Strand cDNA Synthesis Kit l 2 Thaw all reverse transcription reagents from the First Strand cDNA Synthesis Kit Mix reagents well by gently inverting the tubes Centrifuge briefly and place on ice Prepare the RNA primer mix Add the following reagents to an RNase free reaction tube that has been placed on ice The final volume is 13ul Component Volume Final concentratio
7. 4 well block ViiA 7 384 well block om ExProfile Gene qPCR Array User Manual Catalog Array layout 10 11 0 271 2 23 rye fs opn JEIEN IEAEEES 3 44 4 5 56 57 58 59 68 alee 1 82 23 ra ee k mS e A 68 o2 93 salen des z D oo alk co ho oo Teseo fer ae oo CE ETE ER ea 25 2 SERENE REE ra as a8 59 5 sa 15154151 165 70 7 18 18 safes 19517 196 oa 20 201 2021203 20200 1 olen 17 7 2ra 218 220 221 220 TA 229 220 234 232 233 234 235 236 237 K zalaze ae 8 eee e ae zr ze alaso 252 L 7 8 M 298 299 20 201 302 303 306 305 306 310 311 31 N 320 20 321 32 323 28 305 306 27 329320 300 331 32 393 34 335 33 o a7 326 339 340 340 345 24 245 245347 348 240 360361 952 359 256 366 36 257 25813501360 p B Figure 5 Illustration of ExProfile gene qPCR array layout A 96 well plate B 384 well plate O id nm j k at ml Bs k ak j Fe nm amp i on c e FIV Sa anlal Pa HANGAN NNRHAAN o Ma on k oak j ba ERED iO Ca RS fae ae ae ae oO my G S k k k pa co Ma 5 ugo gg in oti a io ar m z oo io io le tae bi S a R x ha i BI Siel oo AA Go co A B C D E F G H J ho a X p 8 ho ni ba IE 1 fr
8. R Mix and cDNA to the qPCR Array Plate C Perform real time PCR or v 7 e e ee ee e ESKE EF 8 SN n n oS an ou n aa Figure 1 Gene qPCR array experiment work flow ExProfile Gene qPCR Array User Manual Performance data Linear Range and Sensitivity spike in control RNA 40 00 y 2 4398x 40 468 35 00 R 0 9962 30 00 25 00 Ct value 20 00 15 00 10 00 0 2 4 6 8 log5 copies Figure 2 Broad linear range and high sensitivity Mouse total RNA with serially diluted Spike in control RNA were reverse transcribed using All in One first strand cDNA synthesis kit The reverse transcribed cDNA samples were detected using All in One qPCR mix and spike in control specific primers deposited in a 96 well plate The resulting Ct values were plotted against the log5 of the amounts of spike in control RNA The data demonstrated a broad linear dynamic range from 4 to 1 6 1 0 copies of input RNA as well as high sensitivity 120 100 80 60 Positive calls 40 20 0 384ng 76 8ng 15 36ng 3 072ng 0 6144ng input RNA in qPCR reaction Figure 3 High positive calls with as little as 15 36 ng of total RNA Different amounts of MCF_7 total RNA 1000 200 40 8 1 6ng were analyzed with the Human Breast Cancer Gene qPCR Array PAG HGBE96 01 The percentage of positive calls Ct lt 35 is plotted against the input amount of total RNA in each qPCR reaction
9. 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 PAG HPKA96 Pathway Related Gene qPCR Array ExProfile Human PI3K AKT Signaling Realted Gene qPCR Array Shipped at room temperate Stable for at least 6 months when stored at 20 C 84 genes PAG HPPK96 1 x 96 well plate 13 ExProfile Gene qPCR Array User Manual ExProfile Human Inflammatory Response and Autoimmunity PAG HPRAQ6 Related Gene qPCR Array ExProfile Human Signal PAG HPST96 Transduction Related Gene qPCR Array ExProfile Human T cell and B cell PAG HPTB96 _ Activation Related Gene qPCR Array ExProfile Human Cell Cycle PAG HPTC96 _ Toxicity and Cancer Related Gene qPCR Array ExProfile Human TGF B Signaling Related Gene qPCR Array PAG HPTG96 PAG HPTH96 ExProfile Human Tumor PAG HPTM96 Metastasis Related Gene qPCR Array ExProfile Hum
10. an Th1 Th2 Th3 Related Gene qPCR Array ExProfile Human T helper 17 Th17 Related Gene qPCR Array PAG HPTS96 TA TM PAG CS ExProfile Custom Gene qPCR Arrays 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate Variable 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 Note New catalog ExProfile gene qPCR arrays will be continuously added to the product line Check out hittp www genecopoeia com product gene qpcr array 14 ExProfile Gene qPCR Array User Manual IX Limited Use License and Warranty Limited use license Following terms and conditions apply to use o
11. 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 ExProfile Gene qPCR Array User Manual ExProfile Human Stomach Cancer Gene qPCR Array ExProfile Human Thyroid Cancer Gene qPCR Array ExProfile Human Testicular Cancer Gene qPCR Array ExProfile Human Cancer Drug Resistance amp Metabolism Related Gene qPCR Array ExProfile Human Cytokine Receptor Related Gene qPCR Array ExProfile Human EGF PDGF Signaling Related Gene qPCR Array ExProfile Human FoxP3 Target Genes gPCR Array ExProfile Human Growth and Development Toxicity Related Gene qPCR Array ExProfile Human Insulin Signaling Related Gene qPCR Array ExProfile Human Innate amp Adaptive Immune Response Related Gene qPCR Array ExProfile Human Inflammatory Cytokines amp Receptors Relayed Gene qPCR Array ExP
12. ene qPCR Array plate is compatible with your qPCR instrument before beginning this protocol 1 Thaw the reagents of All in One 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 2 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 3 Prepare qPCR solution on ice Components 1 well N well 2xAll in One qPCR Mix 10ul 11ul x N cDNA 10 times dilution 1 ul 1 1ul x N 50 X Rox Reference Dye 0 4ul 0 44ulx N ddH2O Not using Rox Reference Dye Qul 9 9ulx N m Using Rox Reference Dye 8 6ul 9 5ulx N Final Volume 20ul 22ulx N a The ExProfile gene qPCR array is used to detect multiple genes 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 4 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 5 Tightly seal the qPCR reaction plate with a new sealing film Ensure that the film seals smoothly to prevent refraction of light and tightly to pr
13. event from evaporation loss Centrifuge briefly in order to remove bubbles 6 Run qPCR The following three step PCR program is recommended for running qPCR Temperature Duration Detection 1 Initial denaturation 95 C 10min No Denaturation 95 C 10sec No 40 Annealing 60 C 20 sec No Extension 72 C 15 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 temperature of the cross linked primer is 60 C when using the optimized All in One qPCR Mix 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 9 ExProfile Gene qPCR Array User Manual Temperature range Heating rate Constant temperature Detection 72 C 95C 0 5 C unit time 10sec unit time Yes 25 C 30 sec No VI 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 up
14. f ExProfile Gene 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
15. ith the reference factor r as follows Nrel NOx NOr N x 27 N x 270 QP OH _ pet 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 Nrel 2 9 220t One e _ AAG The value of 2 samples is the change in expression level of the gene of interest between different Vill Appendix Il Catalog ExProfile gene qPCR array list Shipping and storage Cat No qPCR array products Quantity set Season Shipped at room temperate Stable for at least 6 months when stored at 20 C ExProfile Human Adenocarcinoma 168 genes Gene qPCR Array 2 x 96 well plate PAG HCAD96 11 PAG HCSK96 ExProfile Gene qPCR Array User Manual ExProfile Human Brain Cancer Gene qPCR Array ExProfile Human Breast Cancer Gene qPCR Array ExProfile Human Bladder Cancer Gene qPCR Array ExProfile Human Colorectal Cancer Gene qPCR Array ExProfile Human Cervical Cancer Gene qPCR Array ExProfile Human Endometrial Cancer Gene qPCR Array ExProfile Human Head and Neck Cancer Gene qPCR Array ExProfile Human Kidney Cancer Gene qPCR Array ExProfile Human Leukemia Gene qPCR Array ExProfile Human Lung Cancer Gene qPCR Array ExProfile Human Liver Cancer Gene gPCR Array ExProfile Human Lymphoma Cancer Gene qPCR Array ExProfile Human Myeloma Gene qPCR A
16. n Total RNA 1 ug Spike in control RNA 1 ul 0 08 ng Random primer 250 uM 1 pl 10 uM Oligo dT ig 60 uM 1 ul 2 4 uM ddH20 RNase DNase free to final 13 ul a The final concentration given is recommended Each reaction can contain 10ng to 5yg total RNA Low abundance RNA may not be detected when using less than 10ng total RNA b Using both the Random Primer and the Oligo dT ig primer is recommended for optimal reaction efficiency Mix thoroughly and centrifuge briefly Heat the RNA primer mix from step 2 at 65 C for 10 minutes to denature the RNA After incubation cool immediately on ice then centrifuge briefly Prepare the reverse transcription master mix keeping the tubes from step 3 on ice while working The final volume is 25ul Component Volume Final concentration RNA primer mix 13yl 5 x RT Reaction Buffer Syl 1x dNTP 25mM 1 ul 1mM RNase Inhibitor 25 U ul 1 ul 1U ul M MLV RTase 200U ul 1 ul 8U ul ddH20 RNase DNase free to final 25ul ExProfile Gene qPCR Array User Manual 5 Prepare the reverse transcription reaction by adding the reverse transcription master mix to the RNA primer mixture Mix thoroughly and centrifuge briefly Incubate at 37 C for 60 minutes 6 Terminate the reaction by heating at 85 C for 5 minutes 7 Add 225ul sterile water to each 25ul of reverse transcription reaction 1 10dilution Mix thoroughly and centrifuge briefly qPCR reaction Note Be sure the ExProfile g
17. nd the SYBR Green based All in One qPCR Mix AOPR 0200 AOPR 1000 AOPR 4000 are the designed reagents for use with the ExProfile gene qPCR arrays These reagents have been optimized to produce high sensitivity efficiency and specificity Similar reagents from third party vendors may be compatible with the arrays but not recommended Key advantages Validated primers Each pair of primers for a specific gene is designed using a proprietary algorithm and has been experimentally validated Robust performance Sensitive Detects as low as 4 copies of RNA using ExProfile gene qPCR array and recommended reagents conditions Broad linearity Simultaneously detects mRNAs at different expression levels Reproducible High reproducibility R gt 0 99 for inter array and intra array replicates Genome wide coverage for large selection of catalog and custom arrays Catalog arrays for pathway analysis cancer research and other focused studies Customized arrays for researcher selected gene groups Flexible compatibility Arrays are available in multiple plate formats to ensure compatibility with most commercial RT PCR instruments ExProfile Gene qPCR Array User Manual Convenient data analysis Developed specially for ExProfile arrays a data analysis tool is available for convenient data processing and statistical analysis Protocol overview A Prepare cDNA from your RNA Samples r sample cDNA control cDNA B Add qPC
18. per 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 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 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 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 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 All Ct values reported as greater than 35 or as N A not detected are considered as not detectable QC analysis 1 Examined amplification and melting status of each gene using the qPCR instrument software Each reference gene RI con
19. rofile Human Interferon Signaling amp Response Related Gene qPCR Array ExProfile Human Innate Immune Signaling Related Gene qPCR Array ExProfile Human Interferon Related Gene qPCR Array ExProfile Human JAK STAT Signaling Related Gene qPCR Array ExProfile Human NF amp Kappa amp Beta Signaling 168 genes 2 x 96 well plate 84 genes 1 x 96 well plate 68 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 84 genes 1 x 96 well plate 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
20. rray ExProfile Human Ovarian Cancer Gene qPCR Array ExProfile Human Pancreatic Cancer Gene qPCR Array ExProfile Human Prostate Cancer Gene qPCR Array ExProfile Human Skin Cancer Gene qPCR Array 12 252 genes 3 x 96 well plate 504 genes 6 x 96 well plate 420 genes 5 x 96 well plate 336 genes 4 x 96 well plate 84 genes 1 x 96 well plate 82 genes 1 x 96 well plate 504 genes 6 x 96 well plate 84 genes 1 x 96 well plate 504 genes 6 x 96 well plate 504 genes 6 x 96 well plate 168 genes 2 X 96 well plate 420 genes 5 x 96 well plate 84 genes 1 x 96 well plate 336 genes 4 x 96 well plate 168 genes 2 x 96 well plate 412 genes 5 x 96 well plate 252 genes 3 x 96 well plate 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
21. trol and PCR control should exhibit only one melting peak per reaction Examined CT values of the genomic DNA contamination wells GDC A CT values of genomic DNA control should be more than 35 which indicate a little of genomic DNA contamination can not effect the real time gene expression profiling result Examined CT values of the RT control and 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 RT control should be 2043 and the value of Ct of PCR should be 202 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 qenecopoeia com product gene qpcr array Analysis Tool 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 10 ExProfile Gene qPCR Array User Manual 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 e
22. xperimental 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 of interest to be simply and rapidly selected for further study Vil 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 C 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 w
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