Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity

Abstract

Oncogenic microRNAs (miRs) have emerged as diagnostic biomarkers and novel molecular targets for anti-cancer drug therapies. Real-time quantitative PCR (qPCR) is one of the most powerful techniques for analyzing miRs; however, the use of unsuitable normalizers might bias the results. Tumour heterogeneity makes even more difficult the selection of an adequate endogenous normalizer control. Here, we have evaluated five potential referenced small RNAs (U6, rRNA5s, SNORD44, SNORD24 and hsa-miR-24c-3p) using RedFinder algorisms to perform a stability expression analysis in i) normal colon cells, ii) colon and breast cancer cell lines and iii) cancer stem-like cell subpopulations. We identified SNORD44 as a suitable housekeeping gene for qPCR analysis comparing normal and cancer cells. However, this small nucleolar RNA was not a useful normalizer for cancer stem-like cell subpopulations versus subpopulations without stemness properties. In addition, we show for the first time that hsa-miR-24c-3p is the most stable normalizer for comparing these two subpopulations. Also, we have identified by bioinformatic and qPCR analysis, different miR expression patterns in colon cancer versus non tumour cells using the previously selected suitable normalizers. Our results emphasize the importance of select suitable normalizers to ensure the robustness and reliability of qPCR data for analyzing miR expression.

Identification and validation of circulating miRNAs as endogenous controls in obstructive sleep apnea

“…A seminal study demonstrated the utility of these molecules as biomarkers of adequate response to the treatment of OSA with continuous positive airway pressure (CPAP) [ 8 ]. However, prior to any analysis of circulating miRNAs, normalization is crucial due to the variability that occurs in plasma RNA isolation and analysis [ 14 , 15 ].…”

Identification of miR-29c-3p as a Robust Normalizer for Urine microRNA Studies in Bladder Cancer

“…Several molecules have been proposed as normalizers for miRNAs studies in different disorders, mainly in blood [ 36 ], plasma [ 37 , 38 , 39 ], cell cultures studies [ 25 , 40 ], tissue [ 41 ] and urine [ 35 , 42 , 43 , 44 ]. However, there is no consensus, even in the same sample type, regarding which is the best normalizer for miRNA studies.…”

Impact of long-term storage and freeze-thawing on eight circulating microRNAs in plasma samples

“…The median of UniSp6 was used as a normalization factor to control for sample specific effects on assay performance and to calculate relative quantification levels of miRNAs. The choice of normalization factor was based on the availability of a common molecule in both panels, and its suitability as a reference for data analysis [21,23,[51][52][53].…”