Pc. Goswami et al., A POLYMERASE CHAIN-REACTION ASSAY FOR SIMULTANEOUS DETECTION AND QUANTITATION OF PROTOONCOGENE AND GAPD MESSENGER-RNAS IN DIFFERENT CELL-GROWTH RATES, Cell proliferation, 30(6-7), 1997, pp. 271-282
A reverse transcriptase followed by a polymerase chain-reaction (RT-PC
R) assay was developed for the simultaneous detection and quantitation
of proto-oncogene (c-fos and c-myc) mRNAs using an internal standard
mRNA glyceraldehyde-6-phosphate dehydrogenase (GAPD). Total cellular R
NA was reverse transcribed and PCR amplified with oligonucleotide prim
ers specific to GAPD and either c-fos or c-myc genes. In contrast to N
orthern blot analysis, the RT-PCR assay is rapid and sensitive enough
to quantitate specific proto-oncogene levels from as little as 12-25 n
g of total cellular RNA. The reliability of the assay was tested by me
asuring c-fos and c-myc expression in C3H 10T1/2 mouse embryo fibrobla
st cells under two different growth states: (a) quiescent cell entry i
nto the proliferative cycle, and (b) plateau phase. Furthermore, the a
ssay was used in measuring variations in c-fos or c-myc expression in
HA-1 hamster cells following exposure to the cellular stressing agent,
nitric oxide. In serum-stimulated cells, the RT-PCR measurements of t
ransient increase in c-fos (16-fold at 30 min) and c-myc (10-fold at 1
h) mRNA levels were comparable to previously reported results in the
literature using a Northern blotting assay. In addition, a two-to five
fold increase in c-fos mRNA levels was observed in plateau phase cells
when compared to log phase growth. Furthermore, a transient increase
in c-fos mRNA levels (threefold at 2 h) was also observed following ce
lls' exposure to the stressing agent nitric oxide. These results sugge
st that the multiplex RT-PCR assay represents a significant improvemen
t over current methods to quantitate specific cellular mRNAs under dif
ferent growth conditions or following environmental insults.