A robust method for RNA extraction and purification from a single adult mouse tendon.

Autor:	Grinstein M; Center for Regenerative Medicine, Department of Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA., Dingwall HL; Department of Human Evolutionary Biology, Harvard University, Cambridge, MA, USA., Shah RR; Center for Regenerative Medicine, Department of Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA., Capellini TD; Department of Human Evolutionary Biology, Harvard University, Cambridge, MA, USA.; Broad Institute of Harvard and MIT, Cambridge, MA, USA., Galloway JL; Center for Regenerative Medicine, Department of Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.; Harvard Stem Cell Institute, Cambridge, MA, USA.
Jazyk:	angličtina
Zdroj:	PeerJ [PeerJ] 2018 Apr 24; Vol. 6, pp. e4664. Date of Electronic Publication: 2018 Apr 24 (Print Publication: 2018).
DOI:	10.7717/peerj.4664
Abstrakt:	Background: Mechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts. Methods: Single Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed. Results: After testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons. Discussion: Our work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples. Competing Interests: The authors declare that they have no competing interests.
Databáze:	MEDLINE
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