Selection of Reference Genes for Quantitative RT-PCR Analysis
News Apr 02, 2015
In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or 'housekeeping') gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA.
The article, Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions, is published in PeerJ and is free to access.
Giant Viruses Invent Their Own GenesNews
Three new members have been isolated and added to the Pandoravirus family. This strange family of viruses, with their giant genomes and many genes with no known equivalents, surprised scientists when they were discovered a few years ago. This new study notes that pandoraviruses appear to be factories for new genes – and therefore new functions.
Therapeutic CRISPR Could Be Cancer RiskNews
Therapeutic use of gene editing with the so-called CRISPR-Cas9 technique may inadvertently increase the risk of cancer, according to a new study. Researchers say that more studies are required in order to guarantee the safety of these ‘molecular scissors’ for gene-editing therapies.
Who Owns the Ocean? One Company Has Registered Half of All Marine Gene PatentsNews
A single corporation has registered nearly half of all existing patents associated with genes from marine organisms, according to a new study. Researchers from the University of British Columbia and the Stockholm Resilience Centre examined the patents associated with marine species and found that BASF, the world’s largest chemical manufacturer, has registered 47 per cent of the 12,998 genetic sequences from 862 marine species.