Abstract
BackgroundOne of the most widely used evaluation methods in miRNA experiments is qRT-PCR. However, selecting suitable internal controls (IC) is crucial for qRT-PCR experiments. Currently, there is no consensus on the ICs for miRNA qRT-PCR experiments in breast cancer. To this end, we tried to identify the most stable (the least expression alteration) and promising miRNAs in normal and tumor breast tissues by employing TCGA miRNA-Seq data and then experimentally validated them on fresh clinical samples.MethodsA multi-component scoring system was used which takes into account multiple expression stability criteria as well as correlation with clinical characteristics. Furthermore, we extended the scoring system for more than two biological sub-groups. TCGA BRCA samples were analyzed based on two grouping criteria: Tumor & Normal samples and Tumor subtypes. The top 10 most stable miRNAs were further investigated by differential expression and survival analysis. Then, we examined the expression level of the top scored miRNA (hsa-miR-361-5p) along with two commonly used ICs hsa-miR-16-5p and U48 on 34 pairs of Primary breast tumor and their adjacent normal tissues using qRT-PCR.ResultsAccording to our multi-component scoring system, hsa-miR-361-5p had the highest stability score in both grouping criteria and hsa-miR-16-5p showed significantly lower scores. Based on our qRT-PCR assay, while U48 was the most abundant IC, hsa-miR-361-5p had lower standard deviation and also was the only IC capable of detecting a significant up-regulation of hsa-miR-21-5p in breast tumor tissue.ConclusionsmiRNA-Seq data is a great source to discover stable ICs. Our results demonstrated that hsa-miR-361-5p is a highly stable miRNA in tumor and non-tumor breast tissue and we recommend it as a suitable reference gene for miRNA expression studies in breast cancer. Additionally, although hsa-miR-16-5p is a commonly used IC, it’s not a suitable one for breast cancer studies.
Highlights
Identifying dysregulated genes involved in the carcinogenesis and tumor progression is an important component in cancer research [1]
QRT-PCR internal control for tumor and normal breast tissues recommend it as a suitable reference gene for miRNA expression studies in breast cancer
High-throughput sequencing techniques is being used to conduct the whole transcriptome profiling, the main molecular diagnosis tests in clinic still relies on the cheaper quantitative real-time RT-PCR technique [2]. qRT-PCR is one of the most reliable and powerful tools which promises high specificity, sensitivity, and reproducibility to precisely detect the changes in gene expressions in a broad range of clinical samples, collected under different conditions [3, 4]
Summary
Identifying dysregulated genes involved in the carcinogenesis and tumor progression is an important component in cancer research [1]. The ideal internal control gene is universally valid, with a stable expression level across all tissue samples, cells, and experimental treatments. Such an ideal IC has not yet been found [6, 7]. There is no consensus on the ICs for miRNA qRT-PCR experiments in breast cancer. To this end, we tried to identify the most stable (the least expression alteration) and promising miRNAs in normal and tumor breast tissues by employing TCGA miRNA-Seq data and experimentally validated them on fresh clinical samples
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