Expression Stability Analysis of Candidate References for Normalization of RT-qPCR Data Using RefSeeker R package

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When performing expression analysis either for coding RNA (e.g., mRNA) or non-coding RNA (e.g., miRNA), reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used method. To normalize these data, one or more stable endogenous references must be identified. RefFinder is an online web-based tool using four almost universally used algorithms for assessing candidate endogenous references—delta-Ct, BestKeeper, geNorm, and Normfinder. However, the online interface is presently cumbersome and time consuming. We developed an R package, RefSeeker, which performs easy and straightforward RefFinder analysis by enabling raw data import and calculation of stability from each of the algorithms and provides data output tools to create graphs and tables. This protocol uses RefSeeker R package for fast and simple RefFinder stability analysis.
OriginalsprogEngelsk
Artikelnummere4801
TidsskriftBio-protocol
Vol/bind13
Udgave nummer17
Antal sider14
ISSN2331-8325
DOI
StatusUdgivet - 2023
Eksternt udgivetJa

Bibliografisk note

Funding Information:
EVH received the funding), Danish Cancer Research Foundation: URL:http://www.dansk-kraeftforsknings-fond.dk/ (EVH received the funding), and Herlev Hospital Research Council, URL:

Funding Information:
We thank Douglas Vinicius Oliveira for valuable discussions. Also, we thank Rasmus Adalbert Meldgaard for early testing of the package. We are grateful to the Danish CancerBiobank (Bio- and GenomeBank Denmark) and the Danish Gynecologic Cancer Database for making specimens and data available for use in the present study. This work was founded by: The Mermaid Foundation, URL: http://www.mermaidprojektet.dk/ (PHDP, JLJ, CKH, and EVH received the funding), Danish Cancer Research Foundation: URL:http://www.dansk-kraeftforskningsfond.dk/ (EVH received the funding), and Herlev Hospital Research Council, URL: https://www.herlevhospital.dk/forskning/ (EVH received the funding).

Publisher Copyright:
© 2023 The Authors; exclusive licensee Bio-protocol LLC.

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