Comparison of three methods of RNA extraction from human bone and RNA assessments of osteoarthritic tissue
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Abstract
The comparative analysis of gene expression in human bone can provide insight into underlying molecular mechanisms in Osteoporosis. Identifying comorbidities leading to osteoporosis and other bone metabolic diseases can lead to the discovery of therapeutic targets for human bone disease. There are significant challenges to obtaining transcriptomic data from human bone, including RNA isolation and purification from human bone. In this study, an optimized protocol for obtaining high-quality RNA from human bone samples was established, and transcriptomic data were obtained using RNA assessments to determine comorbidities of osteoporosis across different patient populations.
In this study, three methods of RNA isolation from human bone were compared. To do so, samples from 10 patients undergoing total hip arthroplasty at Boston Medical Center were collected. The three methods compared were hand-grinding human bone, using an automated tissue homogenizer to grind human bone, and 24-hour TRIzol of human bone lysates. The quality of RNA isolated using each method was determined by comparing the total yield of RNA, the purity of the RNA, the RIN value, and qPCR data assessing the average CT value for the 18s ribosomal RNA, COLA1, ALP, SOST, and DMP1 genes. Ultimately, it was found that the optimal protocol for obtaining high-quality RNA from human bone was using the automated tissue homogenizer.
The transcriptomic data in this study was generated using qPCR from high-quality RNA obtained from four patients undergoing total hip arthroplasties. The average CT and relative expression of COLA1, ALP, SOST, and DMP1 compared to 18s were used to determine comorbidities of osteoporosis. The transcriptomic data obtained revealed there are underlying mechanisms, including but not limited to age, sex, and weight for patients undergoing total hip arthroplasties. Future studies should be completed using a larger sample size to determine the underlying molecular mechanisms and comorbidities of osteoporosis.
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Attribution 4.0 International