Key Findings • Affymetrix GeneChip HG-U133 Plus 2.0 microarray data correlates better with VeriQuest Probe qPCR data compared to ABI TaqMan used in the original MAQC study (Fig. 1 and Table 1)
• The location of the real-time PCR primers for each gene has minimal effect on the ability of VeriQuest qPCR Master Mix to validate Affymetrix microarrays (Fig. 1 and Table 1)
• The 84 genes used in this study span a wide range of expression levels (Table 2) and fold-changes between RNA samples (Fig. 1)
• Ready-made IDT PrimeTime qPCR Primers and Probes designed using IDT’s Custom qPCR Design Tool were used for validation demonstrating a robust workflow
Table 1: Average correlation coefficients and average slope values compared to the HG-U133 Plus 2.0 data
qPCR master mix ABI TaqMan® USB VeriQuest® USB VeriQuest®
R
0.93 0.96 0.96
Slope 0.59 0.65 0.66
Notes
qPCR primers mostly towards 5’-end qPCR primers mostly towards 3’-end
Affymetrix microarray data correlates better with VeriQuest qPCR master mix when compared to ABI TaqMan qPCR results. Table summarizes average R and average Slope values for the three sites from Fig. 1.
Table 2: Target genes cover a wide range of expression levels Copies per cell
<1 1-10
10-100 >100
15% 21% 40% 23%
An exogenous control (GeneChip Poly-A controls PN 900433) was used in VeriQuest Probe qPCR master mix experiments to estimate the relative copy numbers for the 84 genes targeted in this study. Results confirm that a wide range of expression levels was used in this experiment.
Conclusion
VeriQuest Probe qPCR Master Mix has better fold-change correlation and slope than the ABI TaqMan Master Mix when comparing to the classic MAQC study using Affymetrix HG-U133 Plus 2.0 arrays. The location of the real-time PCR primers has minimal ef- fect on the fold-change and correlation data. Commercially available primers and design software from IDT yield excellent results. VeriQuest qPCR Master Mixes are the reagents of choice to validate Affymetrix microarray data.
To obtain a sample, or to learn more about VeriQuest qPCR & qRT-PCR master mix validation of microarray data, please visit
usb.affymetrix.com/validate.
Reference: 1. MAQC consortium. (2006) Nature Biotechnology. 24, 1151-1161.
Frequency in MAQC A and B
BioTechniques 58:266-267 (May 2015) doi 10.2144/000114291
Vol. 58 | No. 5 | 2015
267
www.BioTechniques.com
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68