Comparison of methods for isolation of miRNA from bovine milk whey

Abstract Text: Milk miRNAs have great potential to utilize as noninvasive biomarkers for diagnosis or prognosis of diseases due to their high stability in the whey. More than 400 different miRNAs have been found in the mammalian milk. Some protocols have been established for isolation and quantification of miRNAs in whey, but the efficiency and effectiveness of these protocols are variable. The objective of the present study was to compare the methods for isolation of miRNAs from whey. Bovine Milk samples were centrifuged and filtered to obtain the whey, 250 ul of which was lysed by Trizol LS®. Total RNA was isolated from the whey RNA homogenate by two methods: (1) modified phenol based technique (alcohol precipitation with the addition of glycogen as carriers); and (2) column-based clean-up by miRNeasy Mini Kit. Yield and quality of total RNA isolated by these two methods were measured by NanoDrop ND-1000 spectrophotometer. Bioanalyzer 2100 instrument analysis using RNA6000 PicoKit was emplyed for precise detection of the RNA distribution - electropherogram of the microchip gel electrophoresis. The cycle thresholds of several endogenous miRNAs (bta-miR-141, bta-miR-148a, bta-miR-200c and bta-miR-375) and a spike-in synthetic miRNA (cel-miR-39) were tested by RT-qPCR to compare their recovery efficiency between these two methods. Both methods could successfully isolate similar amount of small RNA (<200nt) from whey. Results of cycle thresholds of the endogenous miRNAs and spike-in cel-miR-39 indicated that the column-based clean-up method yielded approximately 10 fold miRNA than the alcohol precipitatio. Nanodrop and bioanalyzer2100 based on RNA6000 PicoKit analysis could not reflect the real miRNA recovery efficiency of the whey. Whereas, spike-in control cel-miR-39 could be utilized as reliable and stable reference due to its perfect performance during the RT-qPCR. In summary, it is preferable to isolate miRNA from whey by combined phenol and column based approach.

Keywords: Isolation, miRNA, whey