Gene qPCR Arrays - tebu-bio
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1. ExProfileTM Gene qPCR Arrays 96 Well 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 ExProfileTM Gene qPCR Array User Manual 2 USER MANUAL ExProfileTM Gene qPCR Array I Introduction II Array Format and Layout III Arrays and Reagents IV Preparation V Procedure VI Data Analysis VII Appendix I VIII Appendix II IX Limited Use License and Warranty I Introduction The ExProfileTM 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 which have been pre validated and coated in designated wells In the same plate there are 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 OneTM First Strand cDNA Synthesis Kits AORT 0020 AORT 0060 and the SYBR Green based All in OneTM qPCR Mix AOPR 0200 AOPR 1000 AOPR 4000 are the designed reagents for use with the ExProfile gene qPCR arrays Thes2. R2 gt 0 99 was also observed for intra array reproducibility data not shown II 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 96 well Applied Biosystems 5700 7000 7300 7500 7700 7900HT Standard 96 well block ViiATM7 Standard 96 well block B 96 well Applied Biosystems 7500 Fast block 7900HT Fast block StepOnePlusTM ViiATM7 Fast block C 96 well Bio Rad Laboratories iCycler iQ MyiQ iQ 5 D 96 well Bio Rad Laboratories CFX96 DNA Engine Opticon DNA Engine Opticon 2 Chromo4 E 96 well Roche Applied Science LightCycler 480 96 well block y 0 9984x 0 1567 R 0 9919 0 00 10 00 20 00 30 00 40 00 0 00 10 00 20 00 30 00 40 00 Ct value of plate B Ct value of plate A Inter array reproducibility ExProfileTM Gene qPCR Array User Manual 6 Catalog Array layout 1 2 3 4 5 6 7 8 9 10 11 12 A 1 2 3 4 5 6 7 8 9 10 11 12 B 13 14 15 16 17 18 19 20 21 22 23 24 C 25 26 27 28 29 30 31 32 33 34 35 36 D 37 38 39 40 41 42 43 44 45 46 47 4
3. Data Analysis System free which is available at http www genecopoeia com product gene qpcr array Analysis_Tool This Data Analysis System uses the Ct method to perform fold change analysis or simple statistical analysis of the expression level Ct or Cp values for each gene 1 Download and read the Primer Array Date Analysis Operation Guide before performing analysis 2 Import the Ct 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 Ct 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 Ct values for the reference factor can be used for normalization ExProfileTM Gene qPCR Array User Manual 11 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 VII Appendix I Ct data analysis method Ct 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 variatio
4. 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 50 Rox 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 l 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 prevent from evaporation loss Centrifuge briefly in order to remove bubbles 6 Run qPCR The following three step PCR program is recommended for running qPCR Cycles Steps Temperature Duration Detection 1 Initial denaturation 95 C 10min a No 40 Denaturation Annealing Extension 95 C 60 Cb 72 Cc 10sec 20 sec 15 sec No No 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 accordin
5. 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 1 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 2 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 13 l Component Volume Final concentration Total RNA 1 ga Spike in control RNA 1ul 0 08ng Random primer 250 M 1 l 10 Mb ddH2O RNase DNase free to final 13 l a The final concentration given is recommended Each reaction can contain 10ng to 5 g total RNA Low abundance RNA may not be detected when using less than 10ng total RNA b The use of Random Primers is recommended for optimal reaction efficiency 3 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 4 Prepare the reverse transcription master mix keeping the tubes from step 3 on ice while working The final volume is 25 l Component Volume Final concentration RNA primer mix 13 l 5 RT Reaction Buffer 5 l 1 dNTP 25mM 1 l 1mM
6. 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 CT 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 analysis 1 Examined amplification and melting status of each gene using the qPCR instrument software Each reference gene RT control and PCR control should exhibit only one melting peak per reaction 2 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 3 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 20 2 and the value of Ct of PCR should be 20 2 across all arrays or samples Data analysis Analyze the qPCR result with GeneCopoeia s online
7. 8 E 49 50 51 52 53 54 55 56 57 58 59 60 F 61 62 63 64 65 66 67 68 69 70 71 72 G 73 74 75 76 77 78 79 80 81 82 83 84 H HK1 HK2 HK3 HK4 HK5 HK6 GDC GDC RT RT PCR PCR Figure 5 Illustration of ExProfileTM gene qPCR array layout 96 well plate RNA primer pairs Wells 1 84 are designated for a real time PCR assay for genes 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 GDC Genomic DNA Controls which can be used to specifically detect genomic DNA contamination with a high level of sensitivity 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 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 III 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 gene qpcr array RT PCR and RNA extraction reagents sold separately Cat No Products Quantity set Shipping and storage condition AORT 0020 AORT 0060 All in OneTM first strand c
8. DNA synthesis kit 20 reactions 60 reactions Shipped with an ice pack Stable for at least 6 months when stored at 20 C AOPR 0200 AOPR 1000 AOPR 4000 All in One qPCR mix 200 reactions 1000 reactions 4000 reactions Shipped with an ice pack Stable for at least 6 months when stored at 20 C E01010A RNAzol RT RNA isolation reagent 50 ml Shipped at room temperature Stable for at least two years when stored at room temperature ExProfileTM Gene qPCR Array User Manual 7 Estimates of number of RT PCR reactions required for EACH SAMPLE Array format Numbers of plates per sample Numbers of RT reactions per sample Numbers of PCR reactions per sample 96 well plate 1 1 110 2 1 220 5 3 550 10 5 1100 20 10 2200 40 20 4400 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 l to 1 000 l adjustable single channel micropipettes with disposable tips 5 l to 20 l 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
9. RNase Inhibitor 25 U l 1 l 1U l M MLV RTase 200U l 1 l 8U l ddH2O RNase DNase free to final 25 l 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 225 l sterile water to each 25 l of reverse transcription reaction 1 10dilution Mix thoroughly and centrifuge briefly ExProfileTM Gene qPCR Array User Manual 9 qPCR reaction Note Be sure the ExProfile gene 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 wella 2 All in One qPCR Mix 10 l 11 l N cDNA 10 times dilution 1 l 1 1 l N 50 X Rox Reference Dyeb 0 4 l 0 44 l N ddH2O Not using Rox Reference Dye Using Rox Reference Dye 9 l 8 6 l 9 9 l N 9 5 l N Final Volume 20 l 22 l N a
10. c 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 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 par
11. e 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 R2 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 ExProfileTM Gene qPCR Array User Manual 3 Convenient data analysis Developed specially for ExProfile arrays a data analysis tool is available for convenient data processing and statistical analysis Protocol overview Figure 1 Gene qPCR array experiment work flow ExProfileTM Gene qPCR Array User Manual 4 Performance data Linear Range and Sensitivity spike in control RNA Figure 2 Broad linear range and hi
12. g 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 72 C 95 C 0 5 C unit time 10sec unit time Yes 25 C 30 sec No ExProfileTM Gene qPCR Array User Manual 10 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 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 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 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
13. gh 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 6 106 copies of input RNA as well as high sensitivity 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 y 2 4398x 40 468 R 0 9962 10 00 15 00 20 00 25 00 30 00 35 00 40 00 0 2 4 6 8 Ct value log5 copies 0 20 40 60 80 100 120 384ng 76 8ng 15 36ng 3 072ng 0 6144ng Positive calls input RNA in qPCR reaction ExProfileTM Gene qPCR Array User Manual 5 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 R2 gt 0 99 was observed for high inter array reproducibility
14. l plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCBA96 ExProfileTM Human Brain Cancer Gene qPCR Arrays 252 genes 3 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCBE96 ExProfileTM Human Breast Cancer Gene qPCR Arrays 504 genes 6 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C ExProfileTM Gene qPCR Array User Manual 12 PAG HCBL96 ExProfileTM Human Bladder Cancer Gene qPCR Arrays 420 genes 5 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCCR96 ExProfileTM Human Colorectal Cancer Gene qPCR Arrays 336 genes 4 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCCV96 ExProfileTM Human Cervical Cancer Gene qPCR Arrays 84 genes 1 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCED96 ExProfileTM Human Endometrial Cancer Gene qPCR Arrays 82 genes 1 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCNN96 ExProfileTM Human Head and Neck Cancer Gene qPCR Arrays 504 genes 6 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCKD96 ExProfileTM Human Kidney Cancer Gene qPCR Arrays 84 genes 1 x 96 well
15. n 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 N0 1 E Ct N0 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 N0 N 2 Ct To analyze the change in expression level for the gene of interest in multiple samples using the Ct 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 N0x N0r N 2 Ctx N 2 Ctr 2 Ctx Ctr 2 Ct 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 Nrel1 Nrel2 2 Ct1 2 Ct2 2 Ct1 Ct2 2 Ct The value of 2 Ct is the change in expression level of the gene of interest between different samples VIII Appendix II Catalog ExProfile gene qPCR array list Cat No qPCR array products Quantity set Shipping and storage condition PAG HCAD96 ExProfileTM Human Adenocarcinoma Gene qPCR Arrays 168 genes 2 x 96 wel
16. plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCLK96 ExProfileTM Human Leukemia Gene qPCR Arrays 504 genes 6 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCLU96 ExProfileTM Human Lung Cancer Gene qPCR Arrays 504 genes 6 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCLV96 ExProfileTM Human Liver Cancer Gene qPCR Arrays 168 genes 2 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCLY96 ExProfileTM Human Lymphoma Cancer Gene qPCR Arrays 420 genes 5 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCML96 ExProfileTM Human Myeloma Gene qPCR Arrays 84 genes 1 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCOV96 ExProfileTM Human Ovarian Cancer Gene qPCR Arrays 336 genes 4 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCPC96 ExProfileTM Human Pancreatic Cancer Gene qPCR Arrays 168 genes 2 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCPS96 ExProfileTM Human Prostate Cancer Gene qPCR Arrays 412 genes 5 x 96 well plate Shipped at room temperate Stable for at least 6 months when sto
17. red at 20 C PAG HCSK96 ExProfileTM Human Skin Cancer Gene qPCR Arrays 252 genes 3 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCSM96 ExProfileTM Human Stomach Cancer Gene qPCR Arrays 168 genes 2 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCTR96 ExProfileTM Human Thyroid Cancer Gene qPCR Arrays 84 genes 1 x 96 well plate Shipped at room temperate Stable for at least 6 months when stored at 20 C PAG HCTT96 ExProfileTM Human Testicular Cancer Gene qPCR Arrays 68 genes 1 x 96 well plate Shipped at room temperate Stable for at least 6 months ExProfileTM Gene qPCR Array User Manual 13 when stored at 20 C Note New catalog ExProfile gene qPCR arrays will be continuously added to the product line Check out http www genecopoeia com product gene qpcr array IX Limited Use License and Warranty Limited use license Following terms and conditions apply to use of ExProfileTM 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 diagnosti
18. ticular 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 ViiATM StepOnePlusTM Life Technologies RNAzol Molecular Research Center Inc NanoDrop Thermo Scientific PAG062613
19. 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 Array format Number of plates per sample Total RNA recommended per sample Number of RT reactions per sample Number of qPCR reactions per sample 96 well plate 2 0 04 2 g 1 220 5 0 12 6 g 3 550 10 0 2 10 g 5 1 100 20 0 4 20 g 10 2 200 40 0 8 40 g 20 4 400 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 dilution 40 g RNA mL to determine the RNA concentration 3 Check the RNA integrity by agarose electrophoresis ExProfileTM Gene qPCR Array User Manual 8 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
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