Comparison of Five Commercial Kits for Total RNA Isolation Including microRNA from Three Bovine Milk Fractions

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 five 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 twelve Holstein cows in mid lactation and separated into fractions (fat, whey and cells) by centrifugation. The whey fraction was further subjected to lyophilisation treatment. Total RNA including miRNA was extracted from each fraction using five 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 DNase treated and the quantity (ng/ml) and quality (RIN) assessed. PCR amplifications covering different lengths of one highly expressed gene (LALBA) and one 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 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, 82.39±9.98) than the other kits (L=55.42±16.20, 19.22±2.42; Z=21.27±3.84, 17.55±3.87; E=30.18±5.46, 43.45±10.71; P=44.85±7.05, 27.51±6.88), respectively. DNA contamination was higher (P < 0.05) in the fat (kits L and Z) and whey (kit L) fractions compared to other kits. Overall RIN in the cell fraction was better for kit L (8.5) compared to other kits. Full length transcripts were detected in fat though fat RIN values (2 to 2.9) were low. Kit Q gave the best overall yield (~90ng/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 to the other kits. From our findings, kits Q and L should be preferred for the isolation of total RNA including miRNA from milk cells while kit Q is the most reliable regardless of the fraction.