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2015 ; 10
(11
): e0141853
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With Reference to Reference Genes: A Systematic Review of Endogenous Controls in
Gene Expression Studies
#MMPMID26555275
Chapman JR
; Waldenström J
PLoS One
2015[]; 10
(11
): e0141853
PMID26555275
show ga
The choice of reference genes that are stably expressed amongst treatment groups
is a crucial step in real-time quantitative PCR gene expression studies. Recent
guidelines have specified that a minimum of two validated reference genes should
be used for normalisation. However, a quantitative review of the literature
showed that the average number of reference genes used across all studies was
1.2. Thus, the vast majority of studies continue to use a single gene, with
?-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being
commonly selected in studies of vertebrate gene expression. Few studies (15%)
tested a panel of potential reference genes for stability of expression before
using them to normalise data. Amongst studies specifically testing reference gene
stability, few found ACTB or GAPDH to be optimal, whereby these genes were
significantly less likely to be chosen when larger panels of potential reference
genes were screened. Fewer reference genes were tested for stability in non-model
organisms, presumably owing to a dearth of available primers in less well
characterised species. Furthermore, the experimental conditions under which
real-time quantitative PCR analyses were conducted had a large influence on the
choice of reference genes, whereby different studies of rat brain tissue showed
different reference genes to be the most stable. These results highlight the
importance of validating the choice of normalising reference genes before
conducting gene expression studies.