Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

Esophageal squamous cell carcinoma (ESCC) is the second and third most common malignancy in Iranian males and females, respectively. Treatment of ESCC is largely ineffective due to lack of detection at early stages of the disease. In recent years, miRNA, a small RNA molecule, has drawn much attention to researchers as a potential biomarker for esophageal cancer. miR-93 and miR-143 are two miRNA molecules reported to be frequently deregulated in various cancers, including prostate, stomach, cervix, and etc. The purpose of this study was to investigate the expression levels of these miRNAs and evaluate their diagnostic and therapeutic potential in esophageal squamous cell carcinoma. In this study, total RNA was extracted from 30 tumor tissues and 30 nontumor tissues of esophageal tumor margins, using RNX-plus solution. After validating the quality and quantity of total RNA, cDNAs of interest were synthesized using microRNA-specific cDNA Synthesis Kit. The expression level of miR-93 and miR-143 was evaluated using quantitative real-time PCR with miRNA-specific primers. Finally, the obtained data was analyzed by SPSS ver.20 software and paired t test was performed to observe the significance of difference between groups. The expression level of miR-93 was significantly increased and of miR-143 was significantly decreased in most of the examined tumor tissues, compared to nontumor tissues. Also, our findings did not detect correlation between mir-93 and mir-143 expressions in regard to stage and grade of the samples. These findings suggest that the deregulation of these miRNAs may play an important role in esophageal squamous cell carcinoma. Both miR-93 and miR-143 might be used as potential biomarkers in esophageal squamous cell carcinoma. However, more studies with large population of samples are necessary.

show abstract

“…By the same token, several studies have also used miR-16 for normalizing their target genes data [66][67][68][69].…”

Section: Discussionmentioning

confidence: 99%

Deregulation of miR-93 and miR-143 in human esophageal cancer

et al. 2015

View full text Add to dashboard Cite

show abstract

“…A total of 10 commonly used candidate reference genes for miRNAs were selected from the reference literature (17)(18)(19)(20). Their primer sequences, amplicon size, and amplification efficiencies are presented in Table I.…”

Section: Rt-qpcr Expression Studies Of Candidate Reference Genesmentioning

confidence: 99%

Identification of suitable reference genes for BDV-infected primary rat hippocampal neurons

Mao

Zhang

Guo

et al. 2016

Molecular Medicine Reports

View full text Add to dashboard Cite

Abstract. Borna disease virus (BDV) is a neurotropic RNA virus that infects the limbic system of mammals and results in behavioral disorders. The hippocampus is a core region in the limbic system, which contributes to memory and learning and is important in the regulation of emotion. However, no validated microRNA housekeeping genes have yet been identified in BDV-infected rat primary hippocampal neurons. Proper normalization is key in accurate miRNA expression analysis. The present study used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to evaluate the expression stability of 10 commonly used reference genes 5S, U6, E2 small nucleolar RNA (snoRNA), in BDV-infected rat hippocampal neurons and non-infected controls across 12 days post-infection. The data was analyzed by four statistical algorithms: geNorm, NormFinder, BestKeeper, and the comparative Δ-Ct method. Subsequently, the most suitable reference genes (miR-101a and U87) and the least suitable (snoRNA) were determined by the RankAggreg package. miR-155 was selected as a standard by which to evaluate the most and least suitable reference genes. When normalized to the most stable reference gene there were significant differences between the two groups. However, when the data were normalized to the less stably expressed gene, the results were not significant. miR-101a was recommended as a suitable reference gene for BDV-infected rat primary hippocampal neurons.

show abstract

“…However, there is no universally applicable gene with invariant expression [10,11], and the expression of historical RGs has been reported to change in numerous tissues and in different study protocols [2,3,5,9,12–14]. …”

Section: Introductionmentioning

confidence: 99%

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

et al. 2017

View full text Add to dashboard Cite

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

show abstract

Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

Cited by 100 publications

References 53 publications

Deregulation of miR-93 and miR-143 in human esophageal cancer

Deregulation of miR-93 and miR-143 in human esophageal cancer

Identification of suitable reference genes for BDV-infected primary rat hippocampal neurons

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

Contact Info

Product

Resources

About