HMBS is the most suitable reference gene for RT‑qPCR in human HCC tissues and blood samples

Abstract

Reverse transcription-quantitative (RT-q) PCR is the most feasible and useful technique for identifying and evaluating cancer biomarkers; however, the method requires suitable reference genes for gene expression analysis. The aim of the present study was to identify the most suitable reference gene for the normalization of relative gene expression in human hepatocellular carcinoma (HCC) tissue and blood samples. First, 14 candidate reference genes were selected through a systematic literature search. The expression levels of these genes ( ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPLP0, RPL13A, SDHA, TBP, TFRC and YWHAZ ) were evaluated using human multistage HCC transcriptome data (dataset GSE114564), which included normal liver (n=15), chronic hepatitis (n=20), liver cirrhosis (n=10), and early (n=18) and advanced HCC (n=45). From the 14 selected genes, five genes, whose expression levels were stable in all liver disease statuses ( ACTB, GAPDH, HMBS, PPIA and RPLP0 ), were further assessed using RT-qPCR in 40 tissues (20 paired healthy tissues and 20 tissues from patients with HCC) and 40 blood samples (20 healthy controls and 20 samples from patients with HCC). BestKeeper statistical algorithms were used to identify the most stable reference genes, of which HMBS was found to be the most stable in both HCC tissues and blood samples. Therefore, the results of the present study suggest HMBS as a promising reference gene for the normalization of relative RT-qPCR techniques in HCC-related studies.

Identification of stable reference genes for quantitative gene expression analysis in the duodenum of meat-type ducks

“…Previous studies have shown that HMBS is the most stable housekeeping gene in human hepatocellular carcinoma tissue, blood samples ( 21 ), and brain tissue ( 22 ). Moreover, studies have found that HMBS has good stability in broilers ( 9 , 23 ), layers ( 24 ), and quails ( 25 ).…”

Identification of immunogenic cell death‑related prognostic signatures in pancreatic cancer

“…The primer sequences were as follows: GAPDH sense, 5′-GCACCGTCAAGGCTGAGAAC-3′ and antisense, 5′-ATGAGGTCCACCACCCTGTTG-3′; NT5E sense, 5′-AGCGAGGACTCCAGCAAGTG-3′ and antisense, 5′-CTTGATCCGACCTTCAACTGCTG-3′. GAPDH was used as an internal reference, and relative mRNA expression was calculated using the 2 −∆∆Cq method ( 12 ).…”

Ethanol induces replication fork stalling and membrane stress in immortalized laryngeal cells

“…Calculation of relative expression was done by the delta-delta Ct method as described before. 98 We used the average of the expression of two housekeeping genes, HPRT 1 and HMBS , 99 as an internal control to normalize the expression values. With A and B two separate samples.…”