Expression Stability Analysis of Candidate References for Normalization of RT-qPCR Data Using RefSeeker R package
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Expression Stability Analysis of Candidate References for Normalization of RT-qPCR Data Using RefSeeker R package. / Petersen, Patrick H.D.; Lopacinska-Jørgensen, Joanna; Høgdall, Claus K.; Høgdall, Estrid V.
In: Bio-protocol, Vol. 13, No. 17, e4801, 2023.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Expression Stability Analysis of Candidate References for Normalization of RT-qPCR Data Using RefSeeker R package
AU - Petersen, Patrick H.D.
AU - Lopacinska-Jørgensen, Joanna
AU - Høgdall, Claus K.
AU - Høgdall, Estrid V.
N1 - Publisher Copyright: © 2023 The Authors; exclusive licensee Bio-protocol LLC.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - Coding and non-coding RNA expression
KW - Expression stability
KW - Normalization
KW - R package
KW - RefFinder
KW - RT-qPCR
U2 - 10.21769/BioProtoc.4801
DO - 10.21769/BioProtoc.4801
M3 - Journal article
C2 - 37719076
AN - SCOPUS:85171547948
VL - 13
JO - Bio-protocol
JF - Bio-protocol
SN - 2331-8325
IS - 17
M1 - e4801
ER -
ID: 396719549