Optimal reference genes for normalization of qPCR gene expression data from proton and photon irradiated dermal fibroblasts

Scientific Reports
Steffen NielsenBrita Singers Sørensen

Abstract

The transcriptional response of cells exposed to proton radiation is not equivalent to the response induced by traditional photon beams. Changes in cellular signalling is most commonly studied using the method Quantitative polymerase chain reaction (qPCR). Stable reference genes must be used to accurately quantify target transcript expression. The study aim was to identify suitable reference genes for normalisation of gene expression levels in normal dermal fibroblasts irradiated with either proton or photon beams. The online tool RefFinder was used to analyse and identify the most stably expressed genes from a panel of 22 gene candidates. To assess the reliability of the identified reference genes, a selection of the most and least stable reference genes was used to normalise target transcripts of interest. Fold change levels varied considerably depending on the used reference gene. The top ranked genes IPO8, PUM1, MRPL19 and PSMC4 produced highly similar target gene expression, while expression using the worst ranked genes, TFRC and HPRT1, was clearly modified due to reference gene instability.

References

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Jun 13, 2008·Nature Protocols·Thomas D Schmittgen, Kenneth J Livak
Oct 8, 2008·Journal of Radiation Research·Nobuyuki Hamada
Jan 17, 2012·International Journal of Radiation Oncology, Biology, Physics·Maarten NiemantsverdrietRobert P Coppes
Oct 8, 2013·Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology·Christian Nicolaj AndreassenJan Alsner
Nov 8, 2013·The British Journal of Radiology·M Durante
Jul 15, 2015·Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology·Brita Singers SørensenJan Alsner
Sep 1, 2015·Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology·Andrea O FontanaMartin N Pruschy
Sep 20, 2015·Cellular Oncology (Dordrecht)·R N SharanMandahakani Ksoo
Nov 18, 2016·Scientific Reports·Rasmus RydbirkTomasz Brudek
Sep 9, 2017·Acta Oncologica·Steffen NielsenBrita Singers Sørensen

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Citations

Sep 23, 2018·Current Treatment Options in Cardiovascular Medicine·Rosalyn O AdigunRekha Mankad
May 15, 2021·Scientific Reports·T TooraniG F Mastromonaco

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Methods Mentioned

BETA
irradiate
biopsies
Assay
PCR

Software Mentioned

HIT12A
RefFinder
BestKeeper
Genorm
Stata
SHIELD
NormFinder

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