Quantitative Reverse Transcriptase Polymerase Chain Reaction for Measuring theN-Methylpurine–DNA Glycosylase mRNA Level in Rodent Cells

Abstract

A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was developed for measuring mRNA concentration, in rodent cells, of theN-methylpurine–DNA glycosylase (MPG), a ubiquitous DNA repair protein responsible for the removal ofN-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 withEcoRI allows separation of the two products because a uniqueEcoRI 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.