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Methods Mol Biol. 2018;1768:401-422. doi: 10.1007/978-1-4939-7778-9_23.

Using Droplet Digital PCR to Analyze Allele-Specific RNA Expression.

Kamitaki N1,2,3, Usher CL1,2,3, McCarroll SA4,5,6.

Author information

1
Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
2
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
3
Department of Genetics, Harvard Medical School, Boston, MA, USA.
4
Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA. mccarroll@genetics.med.harvard.edu.
5
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA. mccarroll@genetics.med.harvard.edu.
6
Department of Genetics, Harvard Medical School, Boston, MA, USA. mccarroll@genetics.med.harvard.edu.

Abstract

Genome-wide association studies have discovered thousands of common alleles that associate with human phenotypes and disease. Many of these variants are in non-protein-coding (regulatory) regions and are believed to affect phenotypes by modifying gene expression. In any organism with a diploid genome, such as humans, measuring the expression of each allele of a gene provides a well-controlled way to identify allelic influences on that gene's expression. Here, we describe a protocol for precisely measuring the allele-specific expression of individual genes. This method targets the nucleotide differences between the two alleles of a gene within an individual and measures the "allelic skew," the extent to which one allele is expressed more than the other. We cover the design of effective assays, the optimization of reactions, and the interpretation of the resulting data.

KEYWORDS:

Allele-specific expression; Allelic imbalance; Allelic skew; Assay design; Digital PCR; Droplet digital PCR; mRNA expression

PMID:
29717456
DOI:
10.1007/978-1-4939-7778-9_23
[Indexed for MEDLINE]

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