Abstract
The expression ratio between the analysed gene and an internal control gene is the most widely used normalization method for quantitative RT-PCR (qRT-PCR) expression analysis. The ideal reference gene for a specific experiment is the one whose expression is not affected by the different experimental conditions tested. In this study, we validate the applicability of five commonly used reference genes during different stages of mouse lung development. The stability of expression of five different reference genes (Tuba1a, Actb Gapdh, Rn18S and Hist4h4) was calculated within five experimental groups using the statistical algorithm of geNorm software. Overall, Tuba1a showed the least variability in expression among the different stages of lung development, while Hist4h4 and Rn18S showed the maximum variability in their expression. Expression analysis of two lung specific markers, surfactant protein C (SftpC) and Clara cell-specific 10 kDA protein (Scgb1a1), normalized to each of the five reference genes tested here, confirmed our results and showed that incorrect reference gene choice can lead to artefacts. Moreover, a combination of two internal controls for normalization of expression analysis during lung development will increase the accuracy and reliability of results.
Highlights
Quantification of transcript levels constitutes one of the crucial aspects of characterizing samples in molecular biology
The ideal reference gene for a specific experiment is the one whose expression is not affected by the different experimental conditions tested
Primer pairs for Tuba1a, Actb and glyceraldehyde phosphate dehydrogenase (Gapdh) were spanning an intron (Figure 1) to avoid artefacts produced by leftovers of genomic DNA or precursor mRNA during quantitative PCR amplification of cDNA after reverse transcription from RNA
Summary
Quantification of transcript levels constitutes one of the crucial aspects of characterizing samples in molecular biology. Techniques to analyse gene expression include reverse transcription followed by polymerase chain reaction (RT-PCR), Northern Blotting, in situ hybridization and microarray-based expression analysis. Use of quantitative PCR to amplify cDNA reverse transcribed from RNA (qRT-PCR) became a routine tool and is the most common technique. RT-PCR is cost-efficient and uncomplicated to perform, allowing rapid throughput. QRT-PCR provides the specificity that is required for accurate and reliable quantification results. QRT-PCR is highly sensitive and permits the detection of rare transcripts and small changes in gene expression. Even a single cell has been used for qRT-PCR based expression analysis [1]
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