Abstract
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has been widely used to quantify relative gene expression because of the specificity, sensitivity, and accuracy of this technique. In order to obtain reliable gene expression data from RT-qPCR experiments, it is important to utilize optimal reference genes for the normalization of target gene expression under varied experimental conditions. Previously, we developed and validated a novel icv-STZ cynomolgus monkey model for Alzheimer’s disease (AD) research. However, in order to enhance the reliability of this disease model, appropriate reference genes must be selected to allow meaningful analysis of the gene expression levels in the icv-STZ cynomolgus monkey brain. In this study, we assessed the expression stability of 9 candidate reference genes in 2 matched-pair brain samples (5 regions) of control cynomolgus monkeys and those who had received intracerebroventricular injection of streptozotocin (icv-STZ). Three well-known analytical programs geNorm, NormFinder, and BestKeeper were used to choose the suitable reference genes from the total sample group, control group, and icv-STZ group. Combination analysis of the 3 different programs clearly indicated that the ideal reference genes are RPS19 and YWHAZ in the total sample group, GAPDH and RPS19 in the control group, and ACTB and GAPDH in the icv-STZ group. Additionally, we validated the normalization accuracy of the most appropriate reference genes (RPS19 and YWHAZ) by comparison with the least stable gene (TBP) using quantification of the APP and MAPT genes in the total sample group. To the best of our knowledge, this research is the first study to identify and validate the appropriate reference genes in cynomolgus monkey brains. These findings provide useful information for future studies involving the expression of target genes in the cynomolgus monkey.
Highlights
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used experimental method for the detection and evaluation of mRNA levels because of the specificity, accuracy, sensitivity, and cost-effectiveness
The reference genes RPS19 and YWHAZ were identified as the 2 most stably expressed genes in the total sample group (Fig. 1A); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and RPS19 were identified in the control group and intracerebroventricular streptozotocin (icv-STZ) group (Fig. 1C and 1E)
Reference genes have been widely analyzed in various species, tissues, and under different experimental conditions; and these reference genes have been widely applied for the accurate quantification of target gene expression levels
Summary
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used experimental method for the detection and evaluation of mRNA levels because of the specificity, accuracy, sensitivity, and cost-effectiveness Despite these advantages, a number of parameters such as differing sample amounts, RNA quality, purity, enzymatic efficiency in reverse transcription, and PCR efficiency can lead to inaccurate quantification of gene expression data by using RT-qPCR experiments [1]. A number of parameters such as differing sample amounts, RNA quality, purity, enzymatic efficiency in reverse transcription, and PCR efficiency can lead to inaccurate quantification of gene expression data by using RT-qPCR experiments [1] To overcome this problem, normalization strategies are commonly used with constitutively expressed gene, termed the reference gene or the internal control gene [2]. Selection of suitable reference genes is important to avoid incorrect results obtained from differential expression patterns in specific tissue types and experimental conditions [4]
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