Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using self-quenched, fluorogenic primers.
Quantitative RT-PCR using LUX primers was performed to determine the expression patterns of various transcripts in samples of pluripotent, mouse P-19 stem cells. The P-19 cells were used because they transform into neuron-like cells upon retinoic acid treatment. The expression of neural and stem cell genes, including GLUR1, GABA-B1a, NMDA1, GAP-43, ChAT, BDNF, nestin, BMP-2, BMP-4, and EGR1, was increased, approximately 10- to 1000-fold, during the course of differentiation from 0 to 11 days after induction with retinoic acid. A 3-fold serial dilution of in vitro-transcribed ChAT mRNA from 66 to 10(7) copies was discriminated by qRT-PCR using fluorogenic LUX primers. Results of quantitation using PCR utilizing dual LUX primer pairs were similar to quantitation using single LUX primers, and to results derived by using an alternate method for qRT-PCR, the 5(')-nuclease probe assay. The efficiencies of PCRs using various primer sets were similar, so that a comparative C(T) method of quantifying relative amounts of transcripts was performed. We conclude that real-time RT-PCR using fluorogenic LUX primers is a reliable, effective alternative to present methods for quantifying several transcripts in neural stem cells.
Lowe
Brian
B
Invitrogen Corporation, Frederick, MD 21704, USA.
Avila
Herbert A
HA
Bloom
Fredric R
FR
Gleeson
Martin
M
Kusser
Wolfgang
W
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D020869:Gene Expression Profiling; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D005456:Fluorescent Dyes; D012333:RNA, Messenger