Evaluation of short-term hair follicle storage conditions for maintenance of RNA integrity

Abstract

Hair follicles provide an easily accessible tissue for interrogating gene expression in mammals. RNAlater is a liquid storage solution that stabilises and preserves cellular RNA, eliminating the need to immediately process or freeze tissue specimens. The manufacturer advises storage of samples at 2-8 degrees C overnight before transfer to -20 degrees C. This study aimed to evaluate RNA integrity in hair follicle samples collected from horses, stabilized in RNAlater, and stored under three short-term storage conditions. Mane hair samples complete with follicles were collected from three horses at a single time point. Approximately 15 hairs were placed in each of three 2 mL tubes containing 0.75ml RNAlater solution. Test group A was stored at 4 degrees C for 24-h, then decanted and stored at -20 degrees C. Test groups B and C were stored at 4 degrees C and 19 degrees C (room temperature) respectively for 7 days, then decanted and stored at -20 degrees C. RNA was isolated from all samples and RNA quantity and quality were measured. One-way ANOVA revealed no difference in RNA concentration (A:516 +/-125 ng/ml, B:273+/-93 ng/ml, C:476+/-176 ng/ml; P = 0.4293) or quality (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.3193) between the test groups. There were no group differences in mean Cycle Threshold values from qPCR validation assays confirming high-quality template cDNA. The results suggest that storage of hair follicles for one week in RNAlater at cool or room temperature conditions will not compromise RNA integrity and will permit extended transport times from remote sampling locations without the need for freezing.