Abstract
Polymerase Chain Reaction (PCR) has become an important diagnostic and research tool of modern molecular biology globally. Real-time PCR allows for rapid and reliable quantification of mRNA transcription. Reference genes are used as internal reaction control to normalise mRNA levels between different samples in order to allow for an exact comparison of mRNA transcription level. In this study, twelve commonly used human reference genes were investigated in Human Embryonic Kidney Cell Lines (HEK293) using real-time qPCR with SYBR green. The genes included beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): UBC, TOP1, ATP5B, CYC1, GAPDH, SDHA, YWHAZ, CTB, 18S, EIFA-2, B2M and RPL13A. The optimal number of reference targets in the experiment was calculated to be 2 (geNorm V<0.15) when comparing a normalization factor based on the 2 or 3 most stable targets). The expression stability varied greatly between the 12 candidate reference genes. UBC, TOP1, ATP5B, CYC1 and GAPDH respectively showed the highest stability in HEK293 cells based on both expression stability and expression level. Overall, our data suggest that UBC and TOP1show the least variation and the highest expression stability. This report validates the need for rational selection of reference genes for data normalization to ensure accuracy of quantitative PCR assays.
Highlights
The polymerase chain reaction (PCR) is a molecular biology based method used for amplifying DNA; for Ribonucleic acid (RNA)-based PCR the RNA sample is first reverse-transcribed to complementary DNA using the reverse transcriptase enzyme [1]
Real-time polymerase chain reaction (RT-qPCR) is a polymerase chain reaction that monitors the amplification of a targeted DNA molecule through monitoring of the fluorescence of dyes or probes introduced into the reaction, which is proportional to the amount of product formed during the cycling phase of the PCR [1]
Gene expression is regulated by cells in all organisms through the turnover of gene and PCR allows for robust detection and quantification of gene expression from small amounts of RNA [2]
Summary
The polymerase chain reaction (PCR) is a molecular biology based method used for amplifying DNA; for RNA-based PCR the RNA sample is first reverse-transcribed to complementary DNA (cDNA) using the reverse transcriptase enzyme [1]. The inclusion of endogenous controls in the assay serves to correct for sample to sample variations It improves the reliability of relative RT-qPCR experiments [3]. Reference genes are used as internal reaction control to normalise mRNA levels between different samples in order to allow for an exact comparison of mRNA transcription level. METHODS: In this study, twelve commonly used human reference genes were investigated in Human Embryonic Kidney Cell Lines (HEK293) using real-time qPCR with SYBR green. The genes included beta-2-microglobulin (B2M), glyceraldehyde-3phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), and tyrosine 3monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes was investigated using the geNorm application. This report validates the need for rational selection of reference genes for data normalization to ensure accuracy of quantitative PCR assays
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