Snapshots of gene expression in rice: limitations for allelic expression imbalance determination

Abstract

In an initial investigation of differential expression of genes caused by cis-acting regulatory elements in rice, the lack of reproducibility led us to question the basic premise of allelic expression imbalance determination: namely that departures of cDNA expression ratios from those observed in genomic DNA provide unequivocal evidence of cis-acting polymorphisms. This paper describes experiments designed to demonstrate that stochastic variation in low copy number of targets in PCR reactions give variable allelic ratios even when starting with the same copy numbers of the two alleles. These significant departures from an expected 1:1 ratio provide an explanation to the lack of reproducibility observed for our cDNA measurements.

Genotyping SSR length variants by isothermal DNA amplification

“…In previous studies quantifying DNA through single molecule amplification (Lee et al 2010;La Mura et al 2011), we know that DNA loss can be considerable during in vitro manipulations if steps are not taken to minimize DNA adhesion to the plasticware. In a previous study with the same DNA sample (Akkinepalli et al 2012), a fivefold difference in DNA concentration was found between values obtained using spectrophotometric means and DNA counting assays: taking the latter value would imply that the 10 Ϫ4 dilution sample represents a single target. The stochastic effect of amplifying from very low numbers of targets (Akkinepalli et al 2012), the amplification of one allele from the heterozygote sample (Fig.…”

“…In a previous study with the same DNA sample (Akkinepalli et al 2012), a fivefold difference in DNA concentration was found between values obtained using spectrophotometric means and DNA counting assays: taking the latter value would imply that the 10 Ϫ4 dilution sample represents a single target. The stochastic effect of amplifying from very low numbers of targets (Akkinepalli et al 2012), the amplification of one allele from the heterozygote sample (Fig. 3) serves to illustrate the low (near single copy) sensitivity of the assay.…”

“…Rice DNA used was obtained from two sources; some samples were obtained from the EURIGEN collection (www.eurigen.net). DNA from the varieties LTH and SHZ and their interspecific F 1 hybrid were extracted from leaves of plants grown in the greenhouse using the CTAB method as described previously (Akkinepalli et al 2012).…”

Biodiversity Assessment of Sugar Beet Species and Its Wild Relatives: Linking Ecological Data with New Genetic Approaches

“…75,76]. Regulatory allelic variants may affect the level of gene expression by causing imbalanced expression of alleles, yet this is a phenomenon poorly studied in plants [77][78][79][80][81]. Identification of co-expressed allelic variants with phenotypic effects can provide novel insights into the biological processes underpinning an adaptive trait [82].…”