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

Mor Grinstein,Rishita R. Shah,Jenna L. Galloway,Terence D. Capellini,Heather L. Dingwall

doi:10.7717/peerj.4664

Mor Grinstein, Rishita R. Shah + Show 3 more

Open Access

https://doi.org/10.7717/peerj.4664

Copy DOI

Abstract

BackgroundMechanistic 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.MethodsSingle 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.ResultsAfter 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.DiscussionOur 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.

Highlights

Tendon injuries are common problems for active individuals and the aging population (Kaux et al, 2011)
Several tissue disruption methods were tested in order to achieve optimal RNA quality and quantity from a single mouse tendon
Obtaining high quality RNA from tendons can be challenging, and this can limit the direction and scope of studies focused on analyzing adult mouse tendon tissues

Summary

Introduction

Tendon injuries are common problems for active individuals and the aging population (Kaux et al, 2011). 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. 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 be applied to other skeletal tissues, as well as precious human samples

Methods

Results

Conclusion