TY - JOUR
T1 - Quantitative reverse transcriptase polymerase chain reaction for measuring the N-methylpurine-DNA glycosylase mRNA level in rodent cells
AU - Roy, Gargi
AU - Roy, Rabindra
AU - Mitra, Sankar
N1 - Funding Information:
1 This research was supported by U.S. Public Health Service Grant CA53791 and NIEHS Center Grant ES07572.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 1997/3/1
Y1 - 1997/3/1
N2 - A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was developed for measuring mRNA concentration, in rodent cells, of the N-methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair protein responsible for the removal of N-alkylpurines and ethenoadducts of adenine, guanine, and cytosine from DNA. The method, applicable for quantitation of any mRNA, is based on the standard approach of comparing the relative amounts of PCR products of the experimental mRNA and a known amount of an exogenous reference RNA which is nearly identical to the experimental RNA. However, unlike in the earlier procedures in which deletion or insertion sequences were added to the reference RNA template, which may affect the efficiency of PCR but are needed to generate different size PCR products, experimental and reference RNAs yield PCR products of the same size in the new method. However, prior digestion with EcoRI allows separation of the two products because a unique EcoRI site was created in the reference RNA vector by point mutations. The QRT-PCR procedure is particularly useful for studying expression of the MPG gene whose mRNA level is very low and difficult to quantitate by Northern blot analysis. The number of MPG mRNA molecules/cell in late log-phase cultures varied from about 6 to 30 in several rodent lines. The SSV-NRK rat cell line has 6 ± 0.2 molecules/cell, while mouse NIH3T3 cells have about 30 ± 1 molecules/cell. If the mRNA level is indicative of the level of the active MPG enzyme, these results may imply a variation in the capacity of various lines to remove the cytotoxic and mutagenic adducts from DNA.
AB - A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was developed for measuring mRNA concentration, in rodent cells, of the N-methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair protein responsible for the removal of N-alkylpurines and ethenoadducts of adenine, guanine, and cytosine from DNA. The method, applicable for quantitation of any mRNA, is based on the standard approach of comparing the relative amounts of PCR products of the experimental mRNA and a known amount of an exogenous reference RNA which is nearly identical to the experimental RNA. However, unlike in the earlier procedures in which deletion or insertion sequences were added to the reference RNA template, which may affect the efficiency of PCR but are needed to generate different size PCR products, experimental and reference RNAs yield PCR products of the same size in the new method. However, prior digestion with EcoRI allows separation of the two products because a unique EcoRI site was created in the reference RNA vector by point mutations. The QRT-PCR procedure is particularly useful for studying expression of the MPG gene whose mRNA level is very low and difficult to quantitate by Northern blot analysis. The number of MPG mRNA molecules/cell in late log-phase cultures varied from about 6 to 30 in several rodent lines. The SSV-NRK rat cell line has 6 ± 0.2 molecules/cell, while mouse NIH3T3 cells have about 30 ± 1 molecules/cell. If the mRNA level is indicative of the level of the active MPG enzyme, these results may imply a variation in the capacity of various lines to remove the cytotoxic and mutagenic adducts from DNA.
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U2 - 10.1006/abio.1996.9992
DO - 10.1006/abio.1996.9992
M3 - Article
C2 - 9056181
AN - SCOPUS:0031106613
VL - 246
SP - 45
EP - 51
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -