Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships. However, the available procedures require multiple steps, are labor intensive and time consuming, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription#150;PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 x 10(11)-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.
Transgenic mice carrying the bacterial lacl gene in a lambda shuttle vector were used to isolate and characterize background and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutations in skin. Adult male mice were treated once topically with either DMBA or acetone or were left untreated. Seven days later, DMBA treatment had significantly increased the mutant frequency in the skin (mean +/- SEM, 36 +/- 3 x 10(-5)) versus in vehicle-treated (6.4 +/- 1.2 x 10(-5)) and untreated mice (7.1 x 1.0 x 10(-5)). At least 10 mutants from each of three DMBA-treated and three untreated mice were selected for DNA sequence analysis. In each case, the entire 1080-bp target gene was sequenced. Base-pair substitutions predominated (86 of 96 mutations), although frameshift and deletion mutations were also detected. Twelve percent of the mutants carried more than one mutation. In controls, the mutations were predominantly GC-->AT transitions (26 of 42), and no AT-->TA transversions were recovered. In contrast, in the DMBA-treated mice, AT-->TA transversions represented 42% of the mutations (23 of 54) and GC-->AT transitions accounted for only 11%. The AT-->TA transversions occurred mostly at 5'-CA sites. This class of mutation has been recovered frequently in ras genes from DMBA-treated mice and probably represents an early event in carcinogenesis (Nelson MA et al., Proc Natl Acad Sci USA 89:6398-6402, 1992). Our present results are consistent with the types of DNA damage induced by DMBA. The observation of different mutant frequencies and spectra in treated and control mice demonstrates the utility of this approach in the study of mutagenesis in vivo.
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