Abstract
BackgroundReal-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a critical tool for evaluating the levels of mRNA transcribed from genes. Reliable RT-qPCR results largely depend on normalization to suitable reference genes. Middle cerebral artery occlusion (MCAO) and oxygen–glucose deprivation/reoxygenation (OGD/R) are models that are commonly used to study ischemic stroke. However, the proper reference genes for RNA analysis in these two models have not yet been determined.ResultsIn this study, we evaluated the expression levels of six candidate housekeeping genes and selected the most suitable reference genes for RT-qPCR analyses of the cortices of MCAO mice and OGD/R-injured N2a cells. Four software programs, geNorm, NormFinder, BestKeeper and RefFinder, were used to validate the stabilities of the candidate reference genes. The results revealed that HPRT and 18S were the most stable reference genes in the cortices of MCAO mice and that β-actin and cyclophilin were the most stable reference genes in the OGD/R-injured N2a cells; in contrast, GAPDH and Sdha were the least stable genes in the cortices of MCAO mice and the OGD/R-injured N2a cells, respectively. Moreover, a combination of HPRT, 18S and cyclophilin was most suitable for normalization in analyses of the cortices of MCAO mice, and a combination of β-actin, cyclophilin, GAPDH, and 18S was most suitable for analyses of the OGD/R-injured N2a cells.ConclusionsThis study provides appropriate reference genes for further RT-qPCR analyses of in vivo and in vitro ischemic stroke and demonstrates the necessity of validating reference genes for RNA analyses under variable conditions.
Highlights
Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a critical tool for evaluating the levels of mRNA transcribed from genes
Specificity and efficiency of the primers The expressional stabilities of the six candidate reference genes in both the Middle cerebral artery occlusion (MCAO) mouse cortex and OGD/Rinjured N2a cells were analyzed by reverse transcribed (RT)-qPCR
Expression of reference genes in the MCAO mouse cortex and oxygen–glucose deprivation/reoxygenation (OGD/R)‐injured N2a cells First, mice were subjected to MCAO for various times (0.5, 1 and 1.5 h) followed by 24 h of reperfusion
Summary
Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a critical tool for evaluating the levels of mRNA transcribed from genes. Reliable RT-qPCR results largely depend on normalization to suitable reference genes. The proper reference genes for RNA analysis in these two models have not yet been determined. Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one of the most important technologies applied to gene mRNA expression analysis in molecular biology. The greatest advantages of RTqPCR are its high sensitivity and ease of operation [1] Some factors, such as the quality of the RNA, the efficiency of the reverse transcription, and the type of. To obtain reliable RT-qPCR results, one must use reference genes to normalize the data. A growing body of evidence indicates that almost every commonly used reference
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