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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.
Highlights
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in adults
actin beta (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and tubulin beta class I (TUBB) were added to the candidate gene list for the step of evaluation
QRT-PCR evaluation quantitative reverse-transcription PCR (qRT-PCR) was carried out to measure the threshold cycle (Ct) values of the candidate genes (Figure 2A), and the results showed that ACTB, TUBB and GAPDH have the highest transcript abundances and ubiquitin conjugating enzyme E2 N (UBE2N) and GAPDH have the largest variations in transcript abundances among the measurements of the nine cell lines with multiple three biological replicates
Summary
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in adults. The molecular mechanisms underlying the initiation and progression of HCC remain elusive; they could most probably result from the changes in the expression levels of several susceptible genes. These cancer-related genes can definitely construct characteristic signal pathways and protein–protein interaction networks, which begin with the occurrence and development of HCC. An important component of studying the HCC mechanisms is detecting the expression pattern in the transcriptome scale through high-throughput profiling assays such as microarray and RNA-seq. These high-throughput results require further validation in most of the circumstances.
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