Comparison of human dental tissue RNA extraction methods for RNA sequencing.

OBJECTIVE: The purpose of this study was to identify an efficient RNA extraction method for periodontal ligament (PDL) and dental pulp (DP) tissues to be used in RNA sequencing studies, given the increased use of these techniques in dental research and the lack of standard protocols.

DESIGN: PDL and DP were harvested from extracted third molars. Total RNA was extracted with four RNA extraction kits. RNA concentration, purity and integrity were assessed by means of NanoDrop and Bioanalyzer and statistically compared.

RESULTS: RNA from PDL was more likely to be degraded than that of DP. The TRIzol method yielded the highest RNA concentration from both tissues. All methods harvested RNA with A260/A280 close to 2.0 and with A260/A230 above 1.5, except for the A260/A230 from PDL obtained with the RNeasy Mini kit. For RNA integrity, the RNeasy Fibrous Tissue Mini kit yielded the highest RIN values and 28 S/18 S from PDL, while the RNeasy Mini kit obtained relatively high RIN values with an appropriate 28 S/18 S for DP.

CONCLUSION: Significantly different results were obtained for PDL and DP when using the RNeasy Mini kit. The RNeasy Mini kit provided the highest RNA yields and quality for DP, while the RNeasy Fibrous Tissue Mini kit obtained the highest quality RNA from PDL.