Selection of internal references for qRT-PCR assays of human hepatocellular carcinoma cell lines

Abstract

Selecting internal references is important for normalizing the loading quantity of samples in quantitative reverse-transcription PCR (qRT-PCR). In the present study, a systematic evaluation of reference genes among nine hepatocellular carcinoma (HCC) cell lines was conducted. After screening the microarray assay data of ten HCC cell lines, 19 candidate reference genes were preselected and then evaluated by qRT-PCR, together with ACTB, GAPDH, HPRT1 and TUBB The expression evenness of these candidate genes was evaluated using RefFinder. The stabilities of the reference genes were further evaluated under different experimental perturbations in Huh-7 and MHCC-97L, and the applicability of the reference genes was assessed by measuring the mRNA expression of CCND1, CCND3, CDK4 and CDK6 under sorafenib treatment in Huh-7. Results showed that TFG and SFRS4 are among the most reliable reference genes, and ACTB ranks third and acts quite well as a classical choice, whereas GAPDH, HPRT1 and TUBB are not proper reference genes in qRT-PCR assays among the HCC cell lines. SFRS4, YWHAB, SFRS4 and CNPY3 are the most stable reference genes of the MHCC-97L under the perturbations of chemotherapy, oxidative stress, starvation and hypoxia respectively, whereas YWHAB is the most stable one of Huh-7 under all perturbations. GAPDH is recommended as a reference gene under chemotherapy perturbations. YWHAB and UBE2B, TMED2 and TSFM, and GAPDH and TSFM are the two best reference genes under oxidative stress, starvation and hypoxia perturbations respectively. TSFM is stable in both cell lines across all the perturbations.

Keywords: ACTB; HCC; RefFinder; SFRS4; TFG; internal references; qRT-PCR.

Figure 1. Results of preselecting the reference candidate genes from the expression microarray datasets

Items including gene (light-blue bubbles) and probe (light-green bubbles) are denoted by bubble charts of (A) all in Supplementary Table S2 and (B) pass the preselection cutoff (CV <0.11, ${\overline{I}}_{\mathrm{i}} >1000$, MFC <0.14). X- and Y-axes are denoted as CV and averaged intensity level respectively. Bubble areas are proportional to MCF values; (C) 10, 15 and 6 of the 19 (gene) candidates were preselected from gene, probe and both levels respectively.

Figure 2. Results of assessing the internal reference genes using qRT-PCR

The sample size of each measurement was three biological replicates multiplied by three technical replicates (n=9). (A) Violin plots of C_t values of the 23 candidate genes arranged from left to right according to the ascending order of S.D. of C_t. White circles show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5-times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme values. (B) Circular assessment of reference candidate genes by RefFinder. The gene with the highest instability score (geometric mean of ranking values) will be excluded in the next calculating cycle until the final two genes win. (C) Final assessments of the internal reference genes by RefFinder and its submethods, such as geNorm, NormFinder, ΔC_t and BestKeeper, compared with the microarray-preselected results. Microarray-preselected results use the CV of intensities from gene (ArrayG) or probe level. The latter can select a probe with the minimum CV (ArrayPCV) or the maximum intensity (ArrayPInt).

Figure 3. Stability evaluation of reference candidate genes in MHCC-97L and Huh-7 under different experimental perturbations

Reference candidate gene performances are denoted by bubble charts under (A) chemotherapy perturbations by cisplatin and sorafenib, (B) oxidative stress by H₂O₂, (C) starvation by low glucose and (D) hypoxia. The sample size of each measurement was three biological replicates multiplied by three technical replicates (n=9). Instability scores from circular assessment of reference candidate genes by RefFinder. The gene with the highest instability score (geometric mean of ranking values) will be excluded in the next calculating cycle until the final two genes win. X- and Y-axes are denoted as ReFinder instability scores from MHCC-97L and Huh-7 respectively. Bubble areas are proportional to ReFinder instability scores from the nine HCC cell lines (Figure 2C). TSFM, the best performing reference gene of both cell lines across all the perturbations, is denoted by red bubbles.

Figure 4. Results of checking the applicability of reference genes under sorafenib treatment in the Huh-7

Dot plot of fold-changes of the four reporter genes under sorafenib treatment that were calculated based on the 23 reference genes. Reference genes arranged from left to right according to the ascending order of performances under chemotherapy perturbations in Huh-7 cells. Circles show the mean values, and error bars show the S.E.M.