Optimization of internal reference genes for qPCR in human pancreatic cancer research

Abstract

Background: Pancreatic cancer (PC) has been becoming a common cancer with high mortality and quantitative real-time polymerase chain reaction (qPCR) is one of the best choices for researching gene expression. Internal reference genes, such as actin beta (ACTB) and glyceraldehyde-3-phosphatide hydrogenase (GAPDH) have long been used in relative quantification analysis. But evidence shows that some internal reference genes expression may vary in different tissues, cell lines and different conditions. The present study aimed to find more stable internal reference gene for qPCR experiment in PC.

Methods: Total RNA of human PC tissues were prepared using TRIZOL reagent. qPCR was performed using FastStart Universal SYBR Green Master to reflects the expression of target genes. Normfinder and geNorm were used to analyze the stability of chosen internal reference genes.

Results: According to the results of NormFinder and geNorm, eukaryotic translation initiation factor 2B subunit alpha (EIF2B1) and importin 8 (IPO8) were the same most stable internal reference genes in PCs and non-neoplastic tissues. In addition, EIF2B1 and IPO8 remained the most stable internal reference genes only in PCs. Using a normalization factor NF2 by geNorm as reference, the normalized GAPDH and ACTB expression levels were obviously up-regulated by 3.29- and 2.23-fold change, meanwhile ribosomal protein S17 (RPS17) were down-regulated by 0.77-fold change in PCs comparing with corresponding adjacent tissues.

Conclusions: The use of the combination of EIF2B1 and IPO8 would provide more stable results in differential expression analysis and prognostic analysis of PC.

Keywords: Pancreatic cancer (PC); internal reference gene; normalization; quantitative real-time polymerase chain reaction (qPCR).

Figure 1

Median Ct values and melt curve analysis of candidate internal reference genes expressed in 16 pairs of histologically verified pancreatic cancers and adjacent non-neoplastic tissues. (A) Variation in 11 internal reference gene expression. Y-axis represents median Ct with interquartile range and X represents genes. (B,C,D,E,F,G,H,I,J,K,L) The specificity of qPCR amplification observed from the plots for 11 internal reference genes. Y-axis represents delta Rn and X represents temperature (°C). Delta Rn values represent the magnitude of the signal generated by the given set of PCR conditions. Sixteen pairs of histologically verified PCs and adjacent non-neoplastic tissues were used.

Figure 2

The evaluation of stabilities of 11 candidate internal reference genes expressed in 16 pairs of histologically verified pancreatic cancers and adjacent non-neoplastic tissues. (A) Output of NormFinder. Y-axis represents the inter-group variance of each gene in two groups of pancreatic cancers and adjacent non-neoplastic samples. Error bars represent the average of the inter-group variances of each gene in two groups. The candidate gene with an inter-group variance as close to zero as possible, and having as small errors bars as possible will be picked by NormFinder. The arrows indicate several internal reference genes with low variability. (B) Average expression stability values (M) of remaining control genes. The gene with the highest M-value was stepwise excluded. (C) Determination of the optimal number of control genes for normalization. Y-axis represents pairwise variation between the normalization factor NFn+1 and NFn. When it first appears that Vn/n+1 >0.15 and at the same time Vn+1/n+2 <0.15, n+1 is the optimal number of reference genes for normalization. (D) Scatterplots of normalization factors between NFn (x-axis) and NFn+1 (y-axis) when it includes an (n+1)th most stable control gene. Vn/n+1 represents pairwise variation (n=2 or 3, r_s = spearman correlation coefficient, r_p = person correlation coefficient). The spearman correlation coefficient (r_s) between NF2 and NF3 was up to 0.987. So we chose the NF2 to replace the NF3 to test the expression of one gene of interest.

Figure 3

The evaluation of stabilities of 11 candidate internal reference genes expressed in 16 pancreatic cancers by geNorm. (A) Average expression stability values (M) of remaining control genes. The gene with the highest M-value was stepwise excluded. (B) Determination of the optimal number of control genes for normalization. Y-axis represents pairwise variation between the normalization factor NFn+1 and NFn. When it first appears that Vn/n+1 >0.15 and at the same time Vn+1/n+2 <0.15, n+1 is the optimal number of reference genes for normalization.

Figure 4

Re-quantification of internal reference genes by the normalization factor NF2. The normalization factor NF2 was calculated by geNorm. The horizontal line segments represent mean fold change. Sixteen pairs of histologically verified PCs and adjacent non-neoplastic tissues were used. Genes with significant P value in paired t-test were labeled with triangular spots.

Figure S1

The expression of ACTB and GAPDH in pancreatic cancers (PCs) and matched adjacent non-neoplastic tissues observed from transcriptional microarrays. The post-normalized matrix data was used. If multiple probes were annotated with the same gene, their mean value was computed to represent expression level of this common gene. Gene with P-value of paired t-test <0.05 was considered as differentially expressed gene. According to the result, ACTB and GAPDH were up-regulated in PCs comparing with non-neoplastic tissues.