Multiplexed RT- PCR for high throughput screening applications.
To increase efficiency of high throughput gene expression profiling, we established a new TaqMan RT-PCR (real-time reverse transcriptase-polymerase chain reaction with internal probes for the quantification of PCR products) method for quantitative mRNA expression analysis. In this procedure, we utilized poly-A mRNA capture plates and validated a multiplexed single tube RT-PCR assay for cell culture applications, including compound testing via gene induction measurement. In the described procedure, all steps including RNA extraction, RT and PCR are performed in the same tube, thus significantly enhancing throughput of this method. Optimization of conditions, including the number of cells necessary for detection of mRNA signal was performed. With a relatively abundant message such as GAPDH, we saw a linear response for all of the concentrations tested, from 10,000 cells to 10 cells. We have also demonstrated multiplexing of different targets within the PCR reactions. In these experiments, we combined VIC-labeled probes for GAPDH with several FAM-labeled probes obtained from Assays On Demand (Applied Biosystems). In the reported experiments, multiplexing did not affect the efficiency of RT-PCR. We also demonstrated the utility of this technology for compound screening applications. The described technology also has the potential to accelerate studies on target and biomarker identification and toxicity assessment in ADMET (absorption, distribution, metabolism, elimination, and toxicity) testing.
Maley
Derrick
D
Johnson & Johnson Pharmaceutical Research & Development, Spring House, PA 19477, USA.
Mei
Jay
J
Lu
Hong
H
Johnson
Dana L
DL
Ilyin
Sergey E
SE
D002452:Cell Count; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D011061:Poly A / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012333:RNA, Messenger; D011061:Poly A; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases