Samaneh Khazaei; Sedigheh Gharbi; Seyed Javad Mowla
Abstract
Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with poor prognosis. In this regard, early diagnosis is of vital importance to cure the tumor in its early stages. Novel cancer diagnostic and therapeutic approaches have been recently introduced based on microRNAs (miRNAs). Also, accurate ...
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Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with poor prognosis. In this regard, early diagnosis is of vital importance to cure the tumor in its early stages. Novel cancer diagnostic and therapeutic approaches have been recently introduced based on microRNAs (miRNAs). Also, accurate normalization using appropriate reference genes is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference genes for miRNA quantification in serum samples of ESCC. In this case and control experimental study, two statistical algorithms including GeNorm and NormFinder were used to evaluate the suitability of miR-16 and 5S rRNA and their geometric mean as reference genes. Then, relative expression of miR-451 and miR-24 were evaluated while different normalizer including miR-16, 5S rRNA and their geometric mean were applied. Both GeNorm and NormFinder analyses showed that geometric mean of miR-16 and 5S rRNA is the most stable reference gene in these samples. Also, our data showed that choosing an inappropriate normalizer could change the relative expression of target genes of miR-451 and miR-24 in ESCC samples which emphasize on the importance of selecting a reliable internal control in expression analyses. We demonstrated that geometric mean of two reference genes could increase the reliability of normalizers and also by using geometric mean as reference gene, relative expression of different target is closer to reality.
Nazila Nouraee; Mohammad Vasei; Shahriar Semnani; Seyed Javad Mowla
Abstract
MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellular
processes, and their disregulations have been linked to several pathologic conditions, mainly cancers.
Determining tissue distribution of miRNAs is a prerequisite for understanding their exact functions ...
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MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellular
processes, and their disregulations have been linked to several pathologic conditions, mainly cancers.
Determining tissue distribution of miRNAs is a prerequisite for understanding their exact functions during
development, tissue homeostasis and abnormality. In situ hybridization is a powerful technique to delineate the
sub-cellular localization and tissue distribution patterns of mRNAs as well as miRNAs. Due to the important
role of miRNAs in tumorigenesis, we optimized an ISH technique for detection of two well-known miRNAs
(miR-302 and miR-21) in formalin-fixed paraffin-embedded (FFPE) tumor samples along with a pluripotent
embryonal carcinoma cell line, NTERA-2 (NT2). After fixation of cells on slides/sectioning of FFPE blocks,
proteinase K digestion, probe concentration, antibody development and light sensitive color reaction were
optimized for both the FFPE samples and cell line. Signals for U6 snRNA, as an internal control, were detected
in the nuclei of the cells. MiR-21 and miR-302 expression was detected in the cytoplasm of FFPE samples of
seminoma carcinoma and in NT2 cell line, respectively. In this study, we optimized ISH for miRNA detection
in FFPE samples and NT2 cell line.
