MiR-361-5p as a promising qRT-PCR internal control for tumor and normal breast tissues

Ghanbari, S ; Sharif University of Technology | 2021

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  1. Type of Document: Article
  2. DOI: 10.1371/journal.pone.0253009
  3. Publisher: Public Library of Science , 2021
  4. Abstract:
  5. Background: One 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. Methods: A 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. Results: According 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. Conclusions: miRNA-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. Copyright © 2021 Ghanbari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
  6. Keywords:
  7. MicroRNA ; MicroRNA 361 5p ; Unclassified drug ; MIRN361 microRNA, human ; Adult ; Breast cancer ; Breast tissue ; Cancer tissue ; Clinical article ; Clinical feature ; Controlled study ; Differential gene expression ; Human ; Multi component scoring system ; Primary tumor ; Real time polymerase chain reaction ; RNA sequencing ; Scoring system ; Survival analysis ; Upregulation ; Validation study ; Breast tumor ; Genetics ; Metabolism ; Pathology ; Reverse transcription polymerase chain reaction ; Standard ; Breast ; Breast Neoplasms ; Female ; Humans ; MicroRNAs ; Middle Aged ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction
  8. Source: PLoS ONE ; Volume 16, Issue 6 June , 2021 ; 19326203 (ISSN)
  9. URL: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0253009