Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

Abstract

Reverse-transcription quantitative PCR (RT-qPCR) is a powerful technique for quantification of gene expression. Extraction of RNA directly from human and large animal intervertebral disc (IVD) tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. IVD is consisted of nucleus pulposus (NP) and annulus fibrosis (AF). We compared the yield and quality of RNA extracted from cultured cells using TRIzol and combined TRIzol-column method (TRIspin) with and without bovine NP tissue added for high proteoglycan content simulation. Higher yields were obtained using the TRIzol method compared to the TRIspin method and the quality of RNA extracted using both methods was comparable. Cryosectioning was by far the most effective homogenization method for the tough bovine NP tissue. The typical modification to TRIzol extraction by the use of high salt solution in a previously reported study for tissue with high proteoglycan was insufficient for bovine NP tissue, where undesirable precipitates were formed during the RNA precipitation step. This necessitates other modifications to the protocol. In our study, the combination of additional phase separation and high salt precipitation was shown to be effective to avoid the formation of the undesirable precipitates. The modified TRIzol method was compared with the TRIspin method and shown to give higher RNA yield which resulted in smaller Ct values in RT-qPCR. In addition, we showed that the cryosectioning method was applicable to bovine muscle, mouse IVD and rat NP tissues. This method of RNA preparation also allows simultaneous preparation of tissue for histological study and quantitative gene expression analysis.