Pyrosequencing for the rapid and efficient quantification of allele-specific expression

Abstract

We have developed a rapid and sensitive quantitative assay for the measurement of individual allelic ratios. This assay minimizes time and labor, the need for special restriction endonuclease enzymes for polymorphic sites, and avoids heteroduplex formation seen with traditional quantitative PCR-based methods. It has improved sensitivity compared to other methods and is capable of distinguishing 1% differences in allelic expression. This assay, termed Pyrosequencing for Imprinted Expression (PIE), involves the use of an intron-crossing PCR primer to generate the first PCR product. We applied the assay to analyze Insulin-like Growth Factor-2 (IGF2) imprinting in both human and mouse prostate tissues.

Keywords: :

• imprinting
• pyrosequencing
• intron-crossing primer
• IGF2
• epigenetic

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by National Institutes of Health, No. 5R01CA097131, (http://grants.nih.gov/grants/)