Abstract
In reverse transcription-quantitative polymerase chain reaction (RT-qPCR) studies, endogenous reference genes are routinely used to normalize the expression of target gene studies. In order to precisely evaluate the relative expression of genes in the cells of mice suffering from Kidney Yang Deficiency Syndrome (KYDS) in response to influenza A virus (IAV) H1N1 using RT-qPCR, it is crucial to identify reliable reference genes. In the present study, 15 candidate reference genes (Actb, β2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhae, 18S rRNA, Rpl13, Ubc, Rpl32, and Ppia) were investigated in lung cells from KYDS mice infected with IAV H1N1. NormFinder, BestKeeper, and GeNorm were used to assess the stability of reference genes. The results were authenticated over extended experimental settings by a group of 10 samples. In the present study, we explored a novel method using dual-gene combinations; the difference in gene expression between the model and normal control groups was statistically analyzed by an independent-samples t-test, and the difference in the mean value between the two groups was compared. A P value > 0.05 and the lowest absolute value of the difference indicated the optimal reference two-gene combination. Four additional host innate immune system-related genes (TLR3, TLR4, TLR7, and RIG-I) were analyzed together with the two treatment datasets to confirm the selected reference genes. Our results indicated that none of these 15 candidate reference genes can be used as reference gene individually for relative quantitative fluorescence PCR analysis; however, the combination of Grcc10 and Ppia, based on the process of calculating the higher P value and lower difference values between groups, was the best choice as a reference gene for the lung tissue samples in KYDS mice infected with IAV. This technique may be applied to promote the selection process of the optimal reference gene in other experiments.
Highlights
At present, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is considered to be a sensitive, accurate, highly specific, and fast measurement of gene expression with absolute or relative quantification [1]
After the Kidney Yang Deficiency Syndrome (KYDS) mice were inoculated through the nose with influenza A virus (IAV) H1N1 or normal saline (NS), the body weight and rectal temperature in the KYDS-virus group were found to be significantly lower compared with those in the normal control group from the 6th to the 7th day (P < 0:01) (Figures 1(a) and 1(b))
The copy numbers of H1N1 M gene RNA were >6:1 × 106/ml, as determined using absolute quantification PCR by the standard curve (Figure 1(d)). These results demonstrated that the KYDS-virus model was successfully constructed
Summary
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is considered to be a sensitive, accurate, highly specific, and fast measurement of gene expression with absolute or relative quantification [1]. Gene expression analyses rely on the use of reference genes, which are internal controls for varied samples and different experimental settings. A number of studies have demonstrated that commonly employed reference genes are unstable, as their transcription is closely regulated in various experimental settings and varies among different tissues. Gapdh is known as a key BioMed Research International enzyme in glycolysis in mammalian cellular organisms, and it is frequently used as a stable reference gene. Its expression is not always constant, in cancer, since the expression of Gapdh changes markedly in rapidly dividing and proliferating tumor cells, and improper reference gene selection may lead to inaccurate results in the target gene expression levels [4, 5]. Potential reference genes must be evaluated for their stability by several algorithms, such as GeNorm [6], which are currently available as part of qBase Plus [7], NormFinder [8], and Best Keeper [9, 10]
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