Validation of common reference genes stability in exosomal mRNA‐isolated from liver and breast cancer cell lines

Abstract

Accurate relative gene expression analysis by reverse transcription‐quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well‐known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes‐isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK‐BR‐3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes‐isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers.

Uncovering the stability status of the reputed reference genes in breast and hepatic cancer cell lines

“…A clear band of PCR products on 1% agarose gel electrophoresis ( Fig 1B ), as well as not seeing primer dimers in the related melt curves, proved the specificity of the designed primers. The efficiency and specificity of these primers except hypoxanthine phosphoribosyltransferase 1 ( HPRT1) , TATA-box binding protein ( TBP ), and actin beta ( ACTB) were also studied in exosomal RNA of hepatic and breast cancer cell lines by our team recently [ 14 ]. However, here we confirmed their efficiency and specificity in cellular RNA of three hepatic and three breast cancer cell lines.…”

“…We have recently reported the expression and stability status of reference genes for exosomal content of the same hepatic cancer cell lines as this study and SKBR3 as breast cancer cell line [ 14 ]. Interestingly, the geNorm showed almost similar stability ranking for cellular and exosomal content, in which B2M and YWHAZ were ranked as two stable reference genes (M value < 1) in Huh7, HepG2, and PLC-PRF5 cell lines.…”

“…PCR efficiency (E) was also provided using LinregPCR version 2017 software. Instead of making serial dilutions and drawing standard curves which may cause errors in PCR efficiency calculation due to common pipetting errors, the LinregPCR software calculates PCR efficiency for individual reaction via calculating the slope of the regression line in an exponential phase of the amplification curve, namely Window-of-Linearity (W-o-L) area, and reports the mean PCR efficiency of samples containing same primer pairs [ 14 , 17 ]. Using the LinregPCR software, the imported raw amplification data of each RT-qPCR run, belong to each reference gene analysis was automatically corrected for baseline adjustment, and at least 3 points were incorporated for the W-o-L area of each sample.…”

“…Reference genes and corresponding primers used in this study were the same selected and designed in our previously published articles [11,[14][15][16]. Evidences considering the usage of these reference genes in hepatic and breast cancer cell lines are presented in Table 1; not exactly the cell lines investigated in this study.…”

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

“…In particular, since exosomes are presently and commonly used to identify biomarkers in cancer, the identification of a suitable reference gene for exosome detection is also required ( 47 ). Gorji-Bahri et al ( 48 ) validated reference genes in pooled cancer exosomes, and Dai et al ( 49 ) revealed that GAPDH, YWHAZ and UBC were the most stably expressed reference genes in exosomal RNA isolated from liver and breast cancer cell lines. However, reference genes in HCC tissues and blood were not evaluated by in vitro experiments and another software analysis to determine stable housekeeping genes.…”