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DNA User Educational Series

DNA User Educational Series. How to Profile the Expression of a Panel of Pathway-specific Genes using PCR Arrays. What are PCR Arrays? Sample Preparation RNA Extraction through the real-time PCR Run Data Analysis

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DNA User Educational Series

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  1. DNA User Educational Series How to Profile the Expression of a Panel of Pathway-specific Genes using PCR Arrays.

  2. What are PCR Arrays? • Sample Preparation RNA Extraction through the real-time PCR Run • Data Analysis Generating CT’s from the SDS Software, using the Super Array Data analysis excel template

  3. Examples of PCR Array Pathways PCR Arrays are designed to profile the expression of a panel of genes relevant to a specific pathway or disease state. There are over 60 different cataloged arrays for human, rat, and mouse in the following broad categories: ApoptosisBiomarkersCancerCell CycleCommon DiseasesCytokine and Inflammatory ResponseExtracellular Matrix and Adhesion MoleculesNeuroscienceSignal TransductionStem Cell and DevelopmentToxicology and Drug Metabolism

  4. PCR Array Layout 84 different Pathway specific or disease-focused SYBR Green primer sets Wells H1 through H5 contain a housekeeping gene panel to normalize PCR Array data. Well H6 contains a Genomic DNA Control (GDC) primer set that specifically detects non-transcribed genomic DNA contamination with a high level of sensitivity. Wells H7 through H9 contain replicate Reverse Transcription Controls (RTC) to test the efficiency of the RT² First Strand Synthesis Kit (C-03) reaction with a primer set to detect template synthesized from the kit's built-in external RNA control. Wells H10 through H12 contain replicate Positive PCR Controls (PPC) to test the efficiency of the polymerase chain reaction itself using a pre-dispensed artificial DNA sequence and the primer set that detects it.

  5. RT² Profiler™ PCR Array Human DNA Damage Signaling Pathway • Apoptosis: ABL1, BRCA1, CIDEA, GADD45A, GADD45G, GML, IHPK3, PCBP4, PDCD8, PPP1R15A, RAD21, TP53, TP73. • Cell Cycle: • Cell Cycle Arrest: CHEK1, CHEK2, DDIT3 (CHOP), GADD45A, GML, GTSE1, HUS1, MAP2K6, MAPK12, PCBP4, PPP1R15A, RAD17, RAD9A, SESN1, ZAK. • Cell Cycle Checkpoint: ATR, BRCA1, FANCG, NBS1, RAD1, RBBP8, SMC1L1, TP53. • DNA Repair: • Damaged DNA Binding: ANKRD17, BRCA1, DDB1, DMC1, ERCC1, FANCG, FEN1, MPG, MSH2, MSH3, N4BP2, NBS1, OGG1, PMS2L3 (PMS2L9), PNKP, RAD1, RAD18, RAD51, RAD51L1, REV1L, SEMA4A, XPA, XPC, XRCC1, XRCC2, XRCC3. • Base-excision Repair: APEX1, MBD4, MPG, MUTYH, NTHL1, OGG1, UNG. • Double-strand Break Repair: CIB1, FEN1, G22P1, KUB3, MRE11A, NBS1, PRKDC, RAD21, RAD50. • Mismatch Repair: ABL1, ANKRD17, EXO1, MLH1, MLH3, MSH2, MSH3, MUTYH, N4BP2, PMS1, PMS2, PMS2L3 (PMS2L9), TP73, TREX1. • Other Genes Related to DNA Repair: APEX2, ATM, ATRX, BTG2, CCNH, CDK7, CRY1, ERCC2 (XPD), GTF2H1, GTF2H2, IGHMBP2, LIG1, MNAT1, PCNA, RPA1, SUMO1.

  6. PCR Array Workflow • Isolate RNA from your samples. • Confirm quality of your RNA. • Prepare cDNA from your RNA samples. • Add cDNA to real-time PCR Master mix and aliquot the mixture across your PCR Array plate. • Perform real-time Thermal Cycling. • Set baseline and threshold and export CT’s. • Analyze changes in gene expression using the SuperArray excel sheet template.

  7. Step 1: RNA Extraction • Cultured Cells • Spin column RNA extraction kit • 2-3 million cells is recommended • DNase treatment over column (2x amount, 2x time) • Tissue Samples • TRIzol RNA extraction • Clean up the RNA a spin column • DNase treatment over column (2x amount, 2x time)

  8. Step 2: Confirm RNA Quality • RNA samples should be suspended in RNase-free water, or RNase-free 10mM Tris buffer pH 8.0 (DO NOT use DEPC-treated water) • A260/A280 = 1.8-2.2 • A260/A230 > 1.5 • Run RNA on an agarose gel or BioAnalyzer chip to test the integrity of the RNA • Test for genomic contamination

  9. Step 3: Prepare cDNA • Use the same amount of total RNA for all samples • Recommended starting amount of RNA: 25ng-1000ng (Can use between 5ng-5ug) • RNA concentrations to deliver total amount of RNA in 9uL

  10. Sensitivity Test performed in DNA Analysis Facility • Lost 7 transcripts when going from 500ng starting RNA to 5 ng starting RNA

  11. Step 4: Set up PCR Array Plates Be sure to purchase the correct plates for our system! • Add cDNA to Real-time SYBR Green Master mix and pipette across plate.

  12. Step 5: Perform Thermal Cycling on real-time Instrument • PCR Array plates are currently being run on our Applied Biosystems 7900HT Sequence Detection System.

  13. Step 6: Set Baseline and Threshold • Set baseline and Threshold in SDS software. • Export CT’s.

  14. Step 7: Analyze Changes in Gene Expression • Download excel template from SuperArray website http://www.superarray.com/PCRArrayPlate.php • Copy and paste CT’s into Excel template

  15. Genomic DNA Contamination • Well H6 contains a Genomic DNA Control (GDC) primer set that specifically detects non-transcribed genomic DNA contamination with a high level of sensitivity. • A value of (CtGDC - CtHKG) greater than or equal to four (4), four (4), or ten (10) for human, mouse, or rat RNA samples indicates a level of genomic DNA contamination low enough to not affect gene expression profiling results. Values less than these numbers indicate that genomic DNA contamination is evident. Re-treat the RNA samples with RNase-free DNase followed by re-purification.

  16. Reverse Transcription Controls • Wells H7 through H9 contain replicate Reverse Transcription Controls (RTC) to test the efficiency of the RT² First Strand Kit (C-03) reaction with a primer set to detect template synthesized from the kit's built-in external RNA control. Any impurities that affect the reverse transcription of the external RNA control also affect the reverse transcription of experimental RNA.A value of (CtRTC - CtPPC) less than five (5) indicates no apparent inhibition. A value greater than five (5) provides evidence of impurities that inhibited the reverse transcription phase of the procedure. Double check the quality, purity, and integrity of the RNA.

  17. Positive PCR Controls • Wells H10 through H12 contain replicate Positive PCR Controls (PPC) to test the efficiency of the polymerase chain reaction itself using a pre-dispensed artificial DNA sequence and the primer set that detects it. Any impurities that affect the positive control PCR amplification also affect amplification of the gene-specific products of interest.Values of CtPPC = 20 +2 on each PCR Array indicate a lack of PCR inhibitors. Higher values and more widely variable values across arrays indicate the presence of inhibitors with different concentrations in or effects on the samples. Double check the quality, purity, and integrity of the RNA samples.

  18. Housekeeping Genes • Wells H1 through H5 contain the species-specific housekeeping gene panel listed in the product information included with each cataloged or custom PCR Array. Use the raw threshold cycle data of these genes to choose an appropriate factor to normalize your PCR Array data, either the Ct value of one housekeeping gene or the average Ct value of up to all five genes. The Ct value of the chosen genes should not differ by more than one (1) cycle across the compared samples.

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