Aai 2007-pcr array-poster
0 likes277 views
This document describes a real-time PCR array for simultaneously evaluating the expression of multiple cytokine mRNAs. The RT2Profiler PCR Array demonstrated high reproducibility, specificity, efficiency, sensitivity, and linear dynamic range. Using this array, 29 genes were found to have at least a 5-fold change in expression between resting and stimulated peripheral blood mononuclear cells after 6 hours of stimulation. The array provides a reliable tool for profiling cytokine pathway gene expression.
Aai 2007-pcr array-poster
- 1. Real-Time PCR Array for Simultaneous Evaluation of Multiple Cytokine mRNA Expression Emi Arikawa, Min You, Jie Wang, Jing-yi Lo, Sean Yu, and Jingping Yang SuperArray Bioscience Corporation, Frederick, MD 21704 Abstract Performance of RT²Profiler™ PCR Array RT² Profiler™ Ct Values 10-25 25-30 30-35 ≥35 Not Detectable Total Ave SD Frequency (%) 0.11 45 0.19 41 0.40 11 0.96 3 0 0 0.20 100 Coefficient of Variance (%) A 75 50 25 0 0-2% 2-4% C 35 CHRNA5 30 Ct 20 1010 108 106 104 102 10 15 10 1 5 copy number 4 5 6 7 8 9 10 11 12 A G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 B G13 G14 G15 G16 G17 G18 G19 G20 G21 G22 G23 G24 C G25 G26 G27 G28 G29 G30 G31 G32 G33 G34 G35 G36 D G37 G38 G39 G40 G41 G42 G43 G44 G45 G46 G47 G48 E G49 G50 G51 G52 G53 G54 G55 G56 G57 G58 G59 Special Features of the RT²Profiler™ PCR Array: RT² Profiler™ G60 F G61 G62 G63 G64 G65 G66 G67 G68 G69 G70 G71 G72 G G73 G74 G75 G76 G77 G78 G79 G80 G81 G82 G83 G84 H HK1 HK2 HK3 HK4 HK5 GDC RTC RTC RTC PPC PPC PPC 1.E+02 1.E+04 1.E+06 1.E+08 Data Analysis Controls (wells H1-H12): Five House Keeping Genes (HKG) primers for raw Ct value data normalization (HK1-HK5) Genomic DNA Contamination (GDC) control primers to detect repetitive non-RNA encoding genomic DNA Reverse Transcription Control (RTC) primers to detect an External RNA Control sequence from SuperArray’s RT2 PCR Array First Strand Kit (C-02) Positive PCR Control (PPC) wells with a pre-dispensed external DNA template and primers to detect it to produce a defined Ct value under proper PCR conditions RT2 Primer Design Criteria Amplicon length 50-210 bp Primer length 19-23 nucleotides GC Content 35 – 65 % Tm 60 – 68 ºC Specificity BLAST versus the entire mRNA Refseq database for the relevant species and the E.Coli DNA database Sequence More than ten thermodynamic criteria set to improve priming efficiency and minimize primer-dimer formation e.g., 3' end stability and no self-complementary structure Our stringent primer design criteria and specially formulated PCR master mixes guarantee specificity and help ensure highly efficient amplification for target genes of interest. In addition, all primer sets on the PCR Array are experimentally validated to insure gene-specific amplification. Data Analysis Method Fold-changes in gene expression are calculated using the ΔΔCt method. For each gene of interest (GOI): ΔCt (Control Replicate 1) = Ct (GOI) – average Ct (HKG) ΔCt (Control Replicate 2) = Ct (GOI) – average Ct (HKG) ΔCt (Control Replicate 3) = Ct (GOI) – average Ct (HKG)… ΔCt (Expt Replicate 1) = Ct (GOI) – average Ct (HKG) Download the PCR Array Data Analysis ΔCt (Expt Replicate 2) = Ct (GOI) – average Ct (HKG) Template at: ΔCt (Expt Replicate 3) = Ct (GOI) – average Ct (HKG)… http://superarray.com/pcrarraydataanalysis.php ΔΔCt (GOI) = average ΔCt (Expt) – average ΔCt (Control) The GOI fold-change from control to experimental group = 2 ^ (-ΔΔCt). The significance of the fold-change in gene expression between the two groups is evaluated by the Student t-test. References 1. MAQC Consortium; Shi, L, et al. The MicroArray Quality Control (MAQC) project shows inter- and intra-platform reproducibility of gene expression measurements. Nature Biotechnology. 2006 Sep; 24(9): 1151-1161. 2. Canales RD, et al. Evaluation of DNA microarray results with quantitative gene expression platforms. Nature Biotechnology. 2006 Sep; 24(9): 1115-1122. CSF2 FAM3B FASLG GDF5 GDF8 GDF9 IFNA1 IFNA2 IFNA4 IFNA5 IL10 IL11 IL12A IL12B IL13 TXLNA IL15 IL16 IL25 IL18 IL19 IL1A IL1B IL1F10 IL1F5 IL1F6 IL1F7 IL1F9 IL2 IL20 IL21 IL22 IL24 IL3 IL4 IL5 IL6 IL8 IL9 TGFA 1 INHA 0 10 20 30 40 TGFB3 Average Ct 120% 100% 80% 60% 20% 0% A Genes HPRT1 RPL13A GAPDH BMP8B CSF1 NODAL PDGFA IL7 LTA LTB TGFB1 TGFB2 TNFSF 11 TNFSF 12 TNFSF 13 TNFSF 13B TNFSF 14 TNFSF 4 CD70 ACTB HGDC RTC RTC RTC PPC PPC Gene Table of Human Common Cytokine PCR Array (APHS-021) TNFSF 8 PPC Figure 6: Identifying Differentially Expressed Genes Between Resting and Stimulated PBMC Resting Using the Common Cytokine RT2 Profiler™ PCR Array Profiler™ B D 40% 1.E+10 TNF B2M INHBA LEFTY2 TNFRSF TNFSF 11B 10 BMP7 The flow chart on the left panel illustrates the design of the present study. Peripheral blood mononuclear cells (PBMC) were treated with or without 50ng/mL PMA+ 1µg/mL ionomycin for 6 or 24 hours. After each incubation period, total RNA was isolated from each preparation, and first strand cDNAs were prepared from 500ng total RNA of each sample using RT2 PCR Array First Strand kit (C-02). Template cDNAs were then characterized in technical triplicates using the Human Common Cytokine PCR Array (APHS-21C) with the RT² SYBR Green / ROX PCR master mix (PA-012) on the ABI 7500 FAST® Real-Time PCR System. The above table shows the layout of the PCR Array. Fold changes in gene expression between the stimulated and resting PBMC RNA were calculated using the ΔΔCt method. The significance of the change in gene expression between the two samples was evaluated by unpaired Student t-test for each gene. The level of statistical significance is set at p<0.005. To validate the results obtained from the PCR Array, the protein level of eight selected cytokines secreted by the PBMC (IL-2, 4, 5, 10, 12, 13, and IFN-γ and TNF-α) was measured using a multiplex cytokine ELISA Array (Human Th1/Th2 Cytokines Multi-Analyte Profiler ELISArray™ (MEH-001A)). 140% Gene Copy Quality-controlled primer sets for a thoroughly researched panel of 84 pathway-focused genes (wells G1-G84) Each assay has been experimentally validated to insure gene-specific amplification. DNA sequencing demonstrated 100% of the PCR products amplified from the correct target genes. BMP6 GDF3 IFNK IL17C IL1F8 Standard curves were constructed for selected assays on the PCR Array using a ten-fold serial dilution of purified DNA as templates. The amplification plot and the standard curve for one example are displayed in Panels A and B, respectively. The RT2 PCR system can detect from one to 1010 copies of template with a linear dynamic range of 10 to 109 copies. Panel C shows the amplification efficiencies and their corresponding 95% confidence intervals (CI) for 200 selected assays performed on the PCR Arrays. In this case, a five-fold serial dilution of DNA template was characterized on individual PCR Arrays. The average amplification efficiency is 99 % with a 95% CI of 90 to 110 %. Figure 3: The RT2Profiler™ PCR Array Demonstrates a High Degree of Specificity Profiler™ B A BMP1 BMP2 BMP3 BMP4 BMP5 BMP6 BMP7 BMP1 BMP2 BMP3 BMP4 BMP6 BMP5 BMP7 BMP15 BMP15 C B 100000.00 Stim ulated 10000.00 A sample of Human Universal Total RNA was characterized on the Human TGFβ / BMP Signaling Pathway PCR Array (APH-035A) using the RT2 RealTime™ SYBR Green / Fluorescein PCR Master Mix (PA-011) on the Bio-Rad iCycler®. After a standard PCR cycling and melting curve program, dissociation curves were obtained (Panel A), and the products were characterized by agarose gel electrophoresis (Panel B). Each reaction yields a single genespecific product of the predicted size. Fold Difference (Stimulated/Resting) 3 BMP5 GDF2 IFNG IL17B 2 Figure 4: Cross Platform Comparisons between Different Technologies Technologies — PCR Arrays Yield Similar Results as TaqMan® 16 1000.00 100.00 10.00 1.00 0.10 0.01 12 y = 0.9941x - 0.5701 12 11 10 8 4 -16 -12 -8 -4 4 8 12 16 -4 TaqMan 0.97 TaqMan -8 -12 Correlation (R) PCR Array 86 genes -16 RT 2 PCR Log2 FC QuantiGene QuantiGene 0.93 StaRT-PCR 0.91 0.90* 0.94* 0.92* RT2 Correlation of fold-change between PCR Arrays and other platforms: PCR Array results were compared with three quantitative assays (the foldTaqMan® Gene Expression Assays from Applied Biosystems, Standardized (Sta)RT-PCRTM from Gene Express, and QuantiGene® from Panomics). A custom PCR Array was produced containing primer sets for ninety (90) genes that overlapped the most with each of the MAQC gene lists. For comparison, data from the other gene expression analysis technologies were obtained from published results1, 2. The concordance of the log2 fold differences between the two RNA samples from the PCR Array and each of the other platforms was individually evaluated by regression analysis. The scatter plot for the comparison with TaqMan is presented above. The purple dashed line on the graph represents a straight line with an ideal slope of 1.0. The solid blue line shows the linear regression data fit. The Pearson correlation coefficients (R) between the PCR Array and all three quantitative assays are listed in the above tables. Correlation (R) values labeled with an asterisk (*) were derived from the published data1,2. Conclusions The RT²Profiler™ PCR Array provides highly reliable qRT-PCR gene expression analyses with exceedingly high reproducibility, specificity, amplification efficiency, sensitivity, and a wide linear dynamic range. The PCR Array System performs as well as TaqMan® PCR and is highly comparable with other quantitative gene expression analysis platforms. It is specifically designed to accelerate the task of simultaneous expression profiling of multiple gene targets belonging to a specific pathway or biological function for any laboratories with a real-time PCR instrument. Reproducibility: Replicate Ct value measurements within 0.20 cycle average standard deviation with an average CV of 0.73%. Wide linear dynamic range and high amplification efficiency: A linear dynamic range from 10-109 template copies with an average amplification efficiency of 99% (with a 95% CI of 90% - 110%). Specificity: Amplification of target gene only. Ability to distinguish different genes from the same family with one-base pair difference. Comparability with TaqMan® PCR: High degree of correlation (R=0.97) in the fold-change results with TaqMan®. Convenience and cost-effectiveness: Simultaneous measurement of 84 pathway-related genes and five housekeeping genes with SYBR® Green based real-time PCR technology using thoroughly tested primer sets, eliminating the time required for optimization of the PCR conditions and the need for those expensive probes with a reporter dye. Using the Human Common Cytokine PCR Array, we identified 29 genes that exhibited at least a 5-fold change in gene expression between resting and PMA/ionomycin stimulated peripheral blood mononuclear cells at 6 h after stimulation. Our data show that changes in cytokine mRNA levels detected by PCR Arrays accurately predict changes in protein levels measured by ELISA. Pathway-focused PCR arrays offer a simple, reliable and sensitive tool for parallel profiling of multiple genes in the cytokine pathway. 9 Resting 8 7 6 Column 5 4 3 2 1 A B C D E F G H Row RNA isolated from resting PBMC or PBMC stimulated with PMA+ionomycin for 6 or 24 hours were characterized on the Human Common Cytokine PCR Array RT2Profiler™ PCR Array (APHS-021C). The 6-h results are presented in Panels A to D. The scatter plot (Panel A) depicts a log transformation plot of the relative expression level of each gene (2^(-ΔCt)) between resting and stimulated PBMC. The pink lines indicate the 5fold change in gene expression threshold. The volcano plot (panel B) depicts the log2 fold changes versus the pvalues from the t-test. Panel C plots the fold changes of each gene as a z-axis displacement from the xy-plane representing the 96-well layout of the PCR Array. Genes that showed at least a five-fold difference in expression between the two samples are listed in the table in panel D. A total of 29 genes had at least a 5-fold change in expression between the stimulated and resting PBMC, with 23 genes having increased expression and 6 genes having decreased expression in stimulated PBMC. As shown in the table, at 24 h, the effects of PMA-ionomycin on genes such as BMPs, CSFs, IFN-γ, IL-1β, IL-6, IL-11, TGF-β and TNF were continuously observed, whereas the effects on other genes such as IL-2, 3, 5, 9, 10, 13, 17 and 22 diminished 24 h after stimulation. Secreted cytokine mRNA expression protein level Fold Change Vs (pg/ml) Untreated Cells 2 TaqMan Log 2 FC 1 0 1.E+00 PCR Efficiency and 95% of CI B BMP4 GDF11 IFNB1 3 Coefficient of Variance Range CHRNA5 BMP3 GDF10 IL17A Figure 2: The PCR Array Demonstrates A Wide Dynamic Range and High Amplification Efficiency High A BMP2 FIGF 4 0 4-6% BMP1 IFNA8 Figure 5: Study Design 5 The two MicroArray Quality Control (MAQC) Reference RNA samples1,2 (Universal Human Reference RNA from Stratagene (Catalog# 740000, Lot# 1130623) and Human Brain Reference RNA from Ambion (Catalog# 6050, Lot# 105P055201A)) were each characterized in six replicate PCR arrays. The table in Panel A lists the average standard deviation for different Ct value ranges as well as the percentage of genes in each group (percent frequency). Panel B charts the frequency of genes exhibiting a coefficient of variation in Ct value determination within a given range. Panel C plots the average coefficient of variance for each average Ct values. 25 What is the RT²Profiler™ PCR Array? RT² Profiler™ Mulitple Cytokine Profiling in Stimulated Peripheral Blood Mononuclear Cells (PBMC) Cells Using a Cytokine-Focused PCR Array Cytokine- Figure 1: The PCR Array Yields Highly Reproducible Ct Values across Replicate Plates B 100 C 6 Frequency (%) Cytokine quantification is an important element in studies of inflammation and immune responses. Quantitative RT-PCR, a rapid and sensitive assay, is the preferred method to quantify cytokine mRNA levels because they are often expressed at low levels. The RT2Profiler™ PCR Array combines the reliable performance of SYBR® Green based qRT-PCR with multi-gene profiling capabilities to simultaneously analyze the expression of a panel of genes from the same pathway. Using PCR Arrays, we have monitored the mRNA levels of 84 different cytokines in human peripheral blood mononuclear cells (PBMC) in response to treatment with 50ng/mL PMA and 1µg/mL ionomycin for up to 24 h. The results identify 23 up-regulated and 6 down-regulated genes (with >5 fold-change & p<0.005) in the stimulated cells when compared to the resting cells at 6 h. At 24 h, the effects of PMA-ionomycin on genes such as BMPs, CSFs, IFN-γ, IL-1β, IL-6, IL-11, TGF-β and TNF are continuously observed, while the effects on other genes such as IL-2, 3, 5, 9, 10, 13, 17 and 22 diminish 24 h after stimulation. To validate these results, the protein level of eight selected cytokines secreted by the PBMC was measured using a multiplex ELISA array. Our data show that changes in cytokine mRNA levels detected by PCR Arrays accurately predict changes in protein levels measured by ELISA. Hence, the PCR Array offers a simple, reliable and sensitive tool for multiple cytokine profiling. Figure 7: Comparison between the Changes in Cellular mRNA Expression and Secreted Protein Levels of Cytokines Expression IL-2 IL-4 IL-5 IL-10 0.00 20.00 24 hr 11190.60 6 hr -2.08 6 hr 24 hr 208.71 12.70 24 hr 1.42 IL4 IL5 6 hr -3.87 100000.0 0 hr 6 hr 24 hr 48 hr IL2 0.0 12917 17390 37355 Time (Hours after Stimulation) 0 hr 6 hr 24 hr 48 hr IL4 19.4 50.7 214.3 170.1 Time (Hours after Stimulation) 6 hr 24 hr 0 hr 48 hr 33.6 183.4 190.8 IL5 13.7 Time (Hours after Stimulation) 14000.0 400000.0 12000.0 10000.0 300000.0 IL10 6 hr 24 hr 48 hr 10.4 44.9 533.2 550.9 Time (Hours after Stimulation) 8000.0 200000.0 300.0 200.0 6000.0 4000.0 100000.0 100.0 0.0 2000.0 0.0 0.0 0 hr 6 hr 24 hr 48 hr 0 0 0 IL12 32.3 Time (Hours after Stimulation) 24 hr 40.00 34.54 TNF Time (Hours after Stimulation) 16000.0 400.0 5.0 6 hr 24 hr 1287.18 IFNG 525.91 Time (Hours after Stimulation) 500000.0 500.0 10.0 0.0 0 hr 24 hr 600.0 15.0 100.0 0.0 6 hr 6 hr 144.6744818 IL13 3961.963846 Time (Hours after Stimulation) 700.0 20.0 200.0 50.0 0.0 0.0 -2.46 800.0 25.0 400.0 50.0 50000.0 30.00 0.00 0 24 hr -4.25 Time (Hours after Stimulation) 300.0 150000.0 1000 6 hr IL12B (p40) 30.0 500.0 100.0 24 hr 35.0 150.0 200000.0 35.00 1500 -5.00 1.14 1.06 IL12A (p35) Time (Hours after Stimulation) 600.0 200.0 100.0 250000.0 250.0 150.0 300000.0 2000 500 24 hr Time (Hours after Stimulation) 200.0 350000.0 40.00 500.00 2500 -4.00 6 hr 62.77 IL10 250.0 400000.0 45.00 1000.00 3000 -3.00 1.00 -20.00 Time (Hours after Stimulation) Time (Hours after Stimulation) TNF-α 1500.00 3500 -2.00 1.05 0.00 0.00 -3.00 6 hr Time (Hours after Stimulation) IFN-γ 4500 4000 40.00 100.00 -2.00 IL2 47820.23 IL-13 0.00 -1.00 1.10 200.00 0.00 -1.00 20000.00 IL-12 60.00 1.00 40000.00 1.15 80.00 300.00 2.00 60000.00 0 hr 6 hr 24 hr 48 hr IL13 21.2 229.5 707.9 753.1 Time (Hours after Stimulation) 0.0 0 hr 6 hr 24 hr 48 hr IFNG 0.5 25300 224912404176 Time (Hours after Stimulation) 0 hr 6 hr 24 hr 48 hr TNFa 38.3 1819 8170 14475 Time (Hours after Stimulation) The effects of PMA-ionomyocin on the secretion of the eight selected cytokines were assessed by multiplex cytokine ELISA. In parallel with the PCR Array results (upper panel), a marked increase in cytokine release (lower panel) was seen for IL-2, 10, 13, and IFN-γ and TNF-α, while only moderate changes were detected for IL-4, 5 and 12. The induction in cytokine secretion by PMA-ionomycin were sustained up to 48 h of stimulation, despite the observation of the subdued mRNA expression for some cytokines such as IL-2, 5, 10 and 13 after 24 h of stimulation.