Optimizations for Identifying Reference Genes in Bone and Cartilage Bioengineering

Abstract

Background：Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is commonly considered as the best-established technique for gene expression assay. However, appropriate reference gene set selection remains the critical and challenging subject for the proper understanding of gene expression pattern. Mixed opinions pertain in how to choose optimal reference gene set according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes was the most feasible for the identification of reference genes in bone and cartilage bioengineering experiments. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme ensuring the stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results：The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed different schemes may have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values and eventual normalization of target genes showed that the different selection schemes have significant effect on the eventual normalization of target genes.Conclusions：Based on the results, the proposed cut-off value of Vn/n+1 under 0.15, according to geNorm algorithm, should be considered with caution, especially in certain tissue types such as skeletal muscle and adipose tissue. Instead, the minimum Vn/n+1 should be used as the cut-off for choosing the optimal reference gene set especially when the stability and variation of candidate reference genes in a specific study are unclear.

Reference gene selection for normalizing gene expression using quantitative real‐time PCR in Haemaphysalis longicornis

“…Therefore, the optimal number of reference genes for the normalization of the target gene expression level was determined by geNorm pairwise variation analysis (Vandesompele et al 2002). In previous studies (Liu et al 2014b;Vandesompele et al 2002;Wan et al 2010;Xiong et al 2021), V n /V n + 1 < 0.15 or V n /V n + 1 < 0.2 have been typically used as cutoff values. However, when the V n / V n + 1 values exceed 0.15 or 0.2, the lowest V n /V n + 1 is generally used to determine the number of reference gene for target gene normalization, regardless of the value (Xiong et al 2021), suggesting that the V n /V n + 1 value can be used flexibly.…”

“…In previous studies (Liu et al 2014b;Vandesompele et al 2002;Wan et al 2010;Xiong et al 2021), V n /V n + 1 < 0.15 or V n /V n + 1 < 0.2 have been typically used as cutoff values. However, when the V n / V n + 1 values exceed 0.15 or 0.2, the lowest V n /V n + 1 is generally used to determine the number of reference gene for target gene normalization, regardless of the value (Xiong et al 2021), suggesting that the V n /V n + 1 value can be used flexibly. In the present study, based on the V n /V n + 1 values qRT-PCR reference gene in H. longicornis obtained, the use of a combination of two genes as the reference gene was suggested for target gene normalization in the tick samples under the conditions of different collections months and SFTSV infection status, whereas three genes were recommended under the conditions of different developmental stages in H. longicornis (Fig.…”

“…2010; Xiong et al . 2021), V n /V n + 1 < 0.15 or V n /V n + 1 < 0.2 have been typically used as cutoff values. However, when the V n /V n + 1 values exceed 0.15 or 0.2, the lowest V n /V n + 1 is generally used to determine the number of reference gene for target gene normalization, regardless of the value (Xiong et al .…”