The crucial choice of reference genes: identification of miR-191-5p for normalization of miRNAs expression in bone marrow mesenchymal stromal cell and HS27a/HS5 cell lines

Abstract

Bone marrow mesenchymal stromal cells (BM-MSCs) have a critical role in tissue regeneration and in the hematopoietic niche due to their differentiation and self-renewal capacities. These mechanisms are finely tuned partly by small non-coding microRNA implicated in post-transcriptional regulation. The easiest way to quantify them is RT-qPCR followed by normalization on validated reference genes (RGs). This study identified appropriate RG for normalization of miRNA expression in BM-MSCs and HS27a and HS5 cell lines in various conditions including normoxia, hypoxia, co-culture, as model for the hematopoietic niche and after induced differentiation as model for regenerative medicine. Six candidates, namely miR-16-5p, miR-34b-3p, miR-103a-3p, miR-191-5p, let-7a-5p and RNU6A were selected and their expression verified by RT-qPCR. Next, a ranking on stability of the RG candidates were performed with two algorithms geNorm and RefFinder and the optimal number of RGs needed to normalize was determined. Our results indicate miR-191-5p as the most stable miRNA in all conditions but also that RNU6a, usually used as RG is the less stable gene. This study demonstrates the interest of rigorously evaluating candidate miRNAs as reference genes and the importance of the normalization process to study the expression of miRNAs in BM-MSCs or derived cell lines.

Figure 1

Strategy used for identification of reference genes. After selection of 6 RG candidates, BM-MSC were cultivated in different conditions: normoxia, hypoxia, co-culture and differentiation. After RNA extraction, the expression of candidates was analysed by RT-qPCR. Those results were analysed by geNorm and RefFinder to rank RG candidate by stability and geNorm was used to determine the optimal number of RGs. The selected RGs for each condition were next validated by studying the relative expression of miR-21-5p. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a CC BY 3.0 License (https://creativecommons.org/licenses/by/3.0/).

Figure 2

Expression of candidate reference genes in all samples. Determination of Ct values for 6 candidates reference genes tested in HS27a, HS5 and primary BM-MSCs cultivated in normoxia (a), hypoxia (b), in primary BM-MSCs after 5 days in culture with KG1a cell lines (c) and primary BM-MSCs after differentiation in adipocytes, osteoblasts and chondrocytes (d).

Figure 3

Evaluation of reference genes across normoxia/hypoxia samples. (a) Average expression stability values of reference candidate genes by geNorm. Candidates are ranked by stability form less stable to more stable. (b) Determination of the optimal number of reference genes for normalization by geNorm. Every bar represents a change in normalization accuracy with the stepwise addition of more reference genes. (c) RefFinder analysis of the candidate’s stability. RCR (Recommended Comprehensive Ranking) is the final ranking based on the ranking of each method. (d) RCR values of reference genes by RefFinder. Reference candidates are ranked by the geometric mean of the ranking (RCR) obtained by RefFinder.

Figure 4

Evaluation of reference genes across co-cultivated samples. (a) Average expression stability values of reference candidate genes by geNorm. Candidates are ranked by stability form less stable to more stable. (b) Determination of the optimal number of reference genes for normalization by geNorm. Each bar represents a change in normalization accuracy with the stepwise addition of more reference genes. (c) RefFinder analysis of the candidate’s stability. RCR (Recommended Comprehensive Ranking) is the final ranking based on the ranking of each method. (d) RCR values of reference genes by RefFinder. Reference candidates are ranked by the geometric mean of the ranking (RCR) obtained by RefFinder.

Figure 5

Evaluation of reference genes across differentiated samples. (a) Average expression stability values of reference candidate genes by geNorm. Candidates are ranked by stability form less stable to more stable. (b) Determination of the optimal number of reference genes for normalization by geNorm. Every bar represents a change in normalization accuracy with the stepwise addition of more reference genes. (c) RefFinder analysis of the candidate’s stability. RCR (Recommended Comprehensive Ranking) is the final ranking based on the ranking of each method. (d) RCR values of reference genes by RefFinder. Reference candidates are ranked by the geometric mean of the ranking (RCR) obtained by RefFinder.

Figure 6

Relative quantification of miR-21-5p in all conditions. For each condition, two references genes have been chosen between the two most stable and the two less stable as previously described. (a) Relative quantification of miR-21 in normoxia and hypoxia condition. Most stable reference genes: miR-16/miR-191, less stable reference genes: miR-34b/RNU6A. Normoxia condition has been chosen as reference condition for normalization. (b) Relative quantification of miR-21 in co-culture condition. Most stable reference genes: miR-34b/miR-191, less stable reference genes: let-7a/RNU6A. BM-MSCs cultivated alone has been chosen as reference condition for normalization. (c) Relative quantification of miR-21 in differentiated experiments with most stable reference genes: let-7a/miR-191 and less stable reference genes: miR-103a/miR-34b. Undifferentiated BM-MSCs has been chosen as reference condition for normalization. *P < 0.05.