F1 hybrids resulting from intercrosses of inbred strains have provided an invaluable tool for the study of imprinting. The hybrids can be used to analyze parent-of-origin differences in expression of any gene, provided sequence differences exist between the two parental alleles. Methods used to detect allele-specific expression include ribonuclease protection assays (1) and allele-specific RNA in situ hybridization (2), as well as a number of reverse transcriptase polymerase chain reaction (RT-PCR)-based assays (see, for example, refs. 3 and 4). We describe here two such assays that are quantitative and require only single base differences between the two alleles. Both assays rely on the amplification of the RNA of interest by RT-PCR using primer sets that flank the sequence polymorphism, a method shown previously to yield amplicons whose allelic ratio is proportional to the ratio in the starting material, regardless of the number of cycles of amplification (5).
We describe here a very sensitive technique for RNA structure analysis and the determination of transcription start sites and demonstrate its use for mapping the start site of the imprinted Snrpn gene in individual hippocampal neurons. The method is adapted from reverse transcription-terminal transferase-dependent PCR (RT-TDPCR) to include amplification of the antisense sequence by in vitro transcription just prior to the final PCR step. The method should be useful for analysis of all genes for which variation in promoter usage and/or differences in RNA secondary structure may be specific to a given cell type or developmental stage.
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