Quantitative RT-PCR-Based Analysis of Allele-Specific Gene Expression

  • Judith Singer-Sam
  • Chunguang Gao
Part of the Methods in Molecular Biology™ book series (MIMB, volume 181)


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 refs. 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).


Reverse Transcriptase Polymerase Chain Reaction Bromphenol Blue Allelic Ratio Disodium Ethylenediaminetetraacetic Acid Moloney Murine Leukemia Virus Reverse Transcriptase 
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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Judith Singer-Sam
    • 1
  • Chunguang Gao
    • 1
  1. 1.Division of BiologyBeckman Research Institute, City of HopeDuarte

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