A Novel Strategy Facilitates Reference Gene Selection by RT-qPCR Analysis in Kidney Yang Deficiency Syndrome Mice Infected with the Influenza A (H1N1) Virus

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.

Figure 1

Assessment of the KYDS-virus model. (a) Whole body weight and (b) rectal temperature were measured after KYDS-virus model establishment for 7 days. (c) Visceral indices of different organs in mice. (d) Absolute quantity of influenza A virus (H1N1) M gene expression was calculated in the model group. ^∗∗P < 0.01, compared with normal control; n = 10 in each group. KYDS: Kidney Yang Deficiency Syndrome.

Figure 2

Mean expression stability (M) evaluated using GeNorm and NormFinder and mean expression stability standard deviation [SD (±CP)] evaluated using BestKeeper. M values and SD values rankings are indicated for mouse lung tissue under different conditions. (a) GeNorm, (b) NormFinder, and (c) BestKeeper.

Figure 3

Relative expression levels of toll-like receptor (TLR)3, TLR4, TLR7, and retinoic acid-inducible gene-I (RIG-I) in lung tissues from normal and model mice were measured by reverse transcription-quantitative polymerase chain reaction and normalized to the indicated reference gene. The fold changes of model (KYDS-virus) mice are indicated next to the bars. The expression levels of normal mice were set at a relative expression of 1 (^∗∗P < 0.01 vs. the normal group; n = 10 in each group, mean ± standard deviation). KYDS: Kidney Yang Deficiency Syndrome.

Figure 4

Diagram of strategy for screening reference gene.

Figure 5

Distribution of quantification cycle (Cq) value and relative quantity for candidate reference genes. Box-whisker plot showing raw Cq values (a) and relative quantity (b) distribution of each reference gene in lung tissue. Boxes represent the quartiles with medians, whereas the maximum and minimum values are represented by whiskers.