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

Authors :
Petersen PHD
Lopacinska-Jørgensen J
Høgdall CK
Høgdall EV
Source :
Bio-protocol [Bio Protoc] 2023 Sep 05; Vol. 13 (17), pp. e4801. Date of Electronic Publication: 2023 Sep 05 (Print Publication: 2023).
Publication Year :
2023

Abstract

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. Key features Perform stability analysis using five algorithms: Normfinder, geNorm, delta-Ct, BestKeeper, and RefFinder. Identification of endogenous references for normalization of RT-qPCR data. Create publication-ready graphs and tables output. Step-by-step guide dialog window for novice R users.<br />Competing Interests: Competing interestsThe authors declare no competing interests.<br /> (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

Details

Language :
English
ISSN :
2331-8325
Volume :
13
Issue :
17
Database :
MEDLINE
Journal :
Bio-protocol
Publication Type :
Academic Journal
Accession number :
37719076
Full Text :
https://doi.org/10.21769/BioProtoc.4801