Real-time RT-PCR normalisation; strategies and considerations

By J. Huggett, K. Dheda, Stephen A. Bustin, Alimuddin Zumla. Genes and Immunity, 2005

1,938 citations recorded by the source corpus.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/b87bb3bddbca098ac1cdace1ab87a042f5d6c18c

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In Molecular Biology

1. Bartłomiej Kozera
2. Marcin Rapacz
3. Pieter Mestdagh
4. A. Jordal
5. Jo Vandesompele
6. Frank Speleman
7. Jiani Qi
8. Xiuyun Zhao

Citations in the corpus

• Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus (2011, neutral): “The reference gene-based normalization corrects for variable starting amounts of RNA and for differences in reverse transcription efficiency, as the references are exposed to the same preparation steps as the gene of interest (Radonić et al., 2004; Bustin, 2005; Huggett et al., 2005; Udvardi et al.,”
• Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon (2005, background): “Not surprisingly, the GAPDH gene was ranked worse in five tissues (liver, head kidney, spleen, brain and thymus), confirming the general skepticism against the use of this gene as reference [7,16,20].”
• Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT‐PCR (2008, background): “However, normalization is necessary to correct the variation in RNA integrity, reverse transcription efficiency, and initial sample amount (Huggett et al., 2005).”
• Reference Gene Selection for Real-Time Quantitative Polymerase Chain Reaction of mRNA Transcript Levels in Chinese Cabbage (Brassica rapa L. ssp. pekinensis) (2010, methodology): “Several statistical approaches have been proposed for evaluating reference genes such as geNorm (Hellemans et al. 2007; Huggett et al. 2005; Vandesompele et al. 2002), Normfinder (Andersen et al. 2004), and BestKeeper (Pfaffl 2001).”
• An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development (2006, neutral): “Data normalization can be problematic and several strategies have been reviewed [9].”
• A novel and universal method for microRNA RT-qPCR data normalization (2009, background): “Several studies report on the use of synthetic RNA or miRNA molecules as spike-in controls for mRNA/miRNA expression data normalization [23-26].”
• Reference genes in real-time PCR (2013, methodology): “Referring to data from reviewing articles and observations for which ones are most often subjected to validation, some examples of commonly used reference genes can be given (Thellin et al. 1999; Jin et al. 2004; Radonić et al. 2004; Huggett et al. 2005; Nicot et al. 2005; Hendriks-Balk et al. 2007;”
• Validation of Candidate Reference Genes for the Accurate Normalization of Real-Time Quantitative RT-PCR Data in Rice During Seed Development (2010, background): “In general, the housekeeping genes, such as those encoding actin (ACT), tubulin (TBL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S), are often chosen for normalization in real-time RT-PCR because of their constant expression levels in living organisms (Huggett et al. 2005).”