Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis
Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metam...
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Published in | Scientific reports Vol. 8; no. 1; p. 496 |
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Main Authors | , , , , |
Format | Journal Article Web Resource |
Language | English |
Published |
London
Nature Publishing Group UK
11.01.2018
Nature Publishing Group |
Subjects | |
Online Access | Get full text |
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Summary: | Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR).
Xenopus laevis
is a popular amphibian model for studying vertebrate embryogenesis and development. Further,
X. laevis
is ideal for studying thyroid signaling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. When using PCR based studies, a primary concern is the choice of reference genes. Commonly used references are
eef1a1
,
odc1
,
rpl8
, and
actnB
, although there is a lack of
ad hoc
reference genes for
X. laevis
. Here, we used previously published RNA-seq data on different
X. laevis
stages and identified the top 14 candidate genes with respect to their expression levels as a function of developmental stage and degree of variation. We further evaluated the stability of these and other candidate genes using RT-qPCR on various stages including the unfertilised eggs, whole embryos during early development and brains during late development. We used four different statistical software packages: deltaCT, geNorm, NormFinder and BestKeeper. We report optimized reference gene pair combinations for studying development (early whole embryos), brains at later stages (metamorphosis and adult), and thyroid signalling. These reference gene pairs are suitable for studying different aspects of
X. laevis
development and organogenesis. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 scopus-id:2-s2.0-85040454811 info:eu-repo/grantAgreement/EC/FP7/607142 |
ISSN: | 2045-2322 2045-2322 |
DOI: | 10.1038/s41598-017-18684-1 |