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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Identification of reference genes for RT-qPCR data normalisation in aging studies.

Lourdes González-Bermúdez¹, Teresa Anglada¹, Anna Genescà¹

¹Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain.

Scientific Reports

|September 29, 2019

Summary

Accurate gene expression analysis in aging research requires stable reference genes. This study identifies optimal reference gene pairs for normalizing reverse transcription quantitative-polymerase chain reaction (RT-qPCR) data across different aging models.

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Area of Science:

Gerontology
Molecular Biology
Biotechnology

Background:

Aging alters gene expression, impacting cellular functions and increasing disease risk.
Stable reference genes are essential for accurate gene expression analysis using RT-qPCR.
Previous studies lack validated reference genes for diverse aging models.

Purpose of the Study:

To evaluate the expression stability of eight commonly used reference genes.
To identify the most stable reference gene pairs across three aging models: oncogene-induced senescence (OIS), in vitro aging, and in vivo aging.
To provide validated reference genes for reliable RT-qPCR normalization in aging research.

Main Methods:

Evaluated eight candidate reference genes across OIS, in vitro, and in vivo aging models.
Utilized NormFinder and geNorm algorithms to assess gene expression stability.
Validated selected reference genes by normalizing expression data of known aging-related genes (CDKN1A, APOD, TFRC).

Main Results:

Identified PUM1 and TBP as the most stable pair for OIS.
Found GUSB and PUM1 to be the most stable pair for in vitro aging.
Determined GUSB and OAZ1 as the most stable pair for in vivo aging.
Validated candidate genes demonstrated reliable normalization of target gene expression.

Conclusions:

Accurate normalization is critical for robust RT-qPCR data in aging studies.
Specific reference gene pairs (PUM1/TBP, GUSB/PUM1, GUSB/OAZ1) are recommended for OIS, in vitro, and in vivo aging, respectively.
This study provides a valuable resource for selecting stable reference genes in future aging research.