Comparison of five commercial kits for total RNA isolation including microRNA from three bovine milk fractions.

Dudemaine, P. L.; Fomenky, B.; Dutoit, A.; Béjanin, L.; Ibeagha-Awemu, E. M.

Many different commercial kits are available for the extraction of total RNA including microRNA (miRNA) from different tissues and cells other than milk. Total RNA isolation from different milk fractions (fat, cells, and whey) is possible, but the challenge is having high yield and quality. The aims of this study were to 1) compare the performance of 5 commercial kits used for the extraction of total RNA including miRNA on bovine milk fractions (fat, whey, and somatic cells) and 2) determine which kit is most suitable for RNA extraction from each milk fraction. Milk samples were collected from 12 Holstein cows in mid lactation and separated into fractions (fat, whey, and cells) by centrifugation. The whey fraction was further subjected to lyophilization treatment. Total RNA including miRNA was extracted from each fraction using 5 different commercial kits E (Exiqon miRCURY RNA Isolation Kit), L (Life Technologies mirVana miRNA Isolation Kit), P (Promega ReliaPrep miRNA Cell and Tissue Miniprep System), Q (Qiagen miRNeasy Mini Kit), and Z (ZymoResearch Direct-Zol RNA MicroPrep). Following RNA extraction, samples were treated with deoxyribonuclease (DNase) and the quantity (ng/mL) and quality (RNA integrity number [RIN]) were assessed. Polymerase chain reaction amplifications covering different lengths of 1 highly expressed gene (LALBA) and 1 lowly expressed gene (FABP3) were done to further verify the integrity of mRNA extracted with kit Q from the fat fraction. Differences between means were calculated with the Student's t-test. Kit Q showed higher (P < 0.05) concentrations of total RNA after DNase treatment from the fat and whey fractions (102.50 ± 24.39 and 82.39 ± 9.98, respectively) than the other kits (55.42 ± 16.20 and 19.22 ± 2.42, respectively, for L; 21.27 ± 3.84 and 17.55 ± 3.87, respectively, for Z; 30.18 ± 5.46 and 43.45 ± 10.71, respectively, for E; and 44.85 ± 7.05 and 27.51 ± 6.88, respectively, for P). Deoxyribonucleic acid contamination was higher (P < 0.05) in the fat (kits L and Z) and whey (kit L) fractions compared with other kits. Overall RIN in the cell fraction was better for kit L (8.5) compared with the other kits. Full length transcripts were detected in fat, although fat RIN values (2 to 2.9) were low. Kit Q gave the best overall yield (approximately 90 ng/mL); meanwhile, kit L gave the best quality (RIN = 8.5) for the cell fraction. Similarly, kit Q and L had expected (better) small RNA pattern following Bioanalyzer results with small RNA Chip in cells, compared with the other kits. From our findings, kits Q and L should be preferred for the isolation of total RNA, including miRNA, from milk cells, whereas kit Q is the most reliable regardless of the fraction.

MICRORNA; WHEY; HOLSTEIN-Friesian cattle

Journal of Animal Science, 2017, Vol 95, p168

0021-8812

Academic Journal

10.2527/asasann.2017.341