Advancement in the Methods for Isolating High Quality RNA From Mouse and Rat Intervertebral Disc.

Abstract

Extracting high-quality RNA from intervertebral disc (IVD) tissues poses a great technical challenge due to the presence of a proteoglycan-rich extracellular matrix and naturally occurring RNases. Current study aims to build on previously reported RNA isolation methods to develop sample preparation and RNA precipitation methods that yield high RNA integrity from mouse and rat IVD tissue sub-types reproducibly. High salt-isopropanol RNA precipitation was tested on three sample types. (1) Freshly dissected mouse IVD tissues: nucleus pulposus (NP) and annulus fibrosus (AF) tissues from mouse lumbar and caudal IVD tissues were collected in <i>RNAlater</i> and stored in TRIzol at -80°C. (2) Freshly dissected rat IVD tissues: NP and AF tissues from freshly dissected rat caudal IVD tissues were snap frozen or stored in <i>RNAlater</i> at -80°C. (3) Cultured rat IVD tissues stored at -80°C for > 1 year: long-term stored rat caudal samples were thawed in <i>RNAlater-ICE</i> solution. RNA isolated from freshly dissected mouse lumbar and caudal NP tissues yielded a mean RNA integrity number (RIN) of 9.6 and 9.8, respectively. Mouse lumbar and caudal AF tissues yielded a mean RIN of 8.9 and 8.3, respectively. Snap frozen NP and AF rat tissues yielded a mean RIN of 9.1 and 7.4, respectively. NP and AF tissues stored in <i>RNAlater</i> yielded a mean RIN of 9.6 and 8.3, respectively. Addition of <i>RNAlater-ICE</i> to banked rat IVD samples yielded a mean RIN of 8.2 and 7.6 for NP and AF tissues, respectively. This study describes methods that reproducibly generate high-quality RNA from freshly dissected and stored IVD tissues from mice and rats. Notably, we highlight a method using <i>RNAlater-ICE</i> to yield high-quality RNA from banked rat IVD tissues. These methods will ensure reproducible molecular assessment of rodent IVD tissues to improve our understanding of IVD biology.