Unstable expression of commonly used reference genes in rat pancreatic islets early after isolation affects results of Gene Expression Studies

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00023001%3A_____%2F16%3A00059762" target="_blank" >RIV/00023001:_____/16:00059762 - isvavai.cz</a>

Alternative codes found

RIV/00216208:11110/16:10325281

Result on the web

<a href="http://journals.plos.org/plosone/article/asset?id=10.1371%2Fjournal.pone.0152664.PDF" target="_blank" >http://journals.plos.org/plosone/article/asset?id=10.1371%2Fjournal.pone.0152664.PDF</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1371/journal.pone.0152664" target="_blank" >10.1371/journal.pone.0152664</a>

Result language

angličtina

Original language name

Unstable expression of commonly used reference genes in rat pancreatic islets early after isolation affects results of Gene Expression Studies

Original language description

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

Czech name

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Czech description

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Type

J_x - Unclassified - Peer-reviewed scientific article (Jimp, Jsc and Jost)

CEP classification

FJ - Surgery including transplantology

OECD FORD branch

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Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year

2016

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

PLoS ONE [online]

ISSN

1932-6203

e-ISSN

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Volume of the periodical

11

Issue of the periodical within the volume

4

Country of publishing house

US - UNITED STATES

Number of pages

18

Pages from-to

"e0152664"

UT code for WoS article

000373201200029

EID of the result in the Scopus database

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