Optimizing miR-29 measurements in biobanked, heparinized samples

Abstract

Aims: MicroRNAs (miRs) and their importance in development, normal physiology, and disease have become increasingly recognized. Our laboratory is interested in miR-29 and its effects on lung development. These studies set out to identify optimal conditions for the measurement of miR-29 in heparinized, biobanked samples and to compare isoform expression patterns.

Materials and methods: The efficiency of three distinct heparinases were tested using reverse transcriptase polymerase chain reaction (RT-PCR): recombinant F. Heparinum heparinase I; recombinant P. heparinus heparinase II; recombinant P. heparinus heparinase III; and heparinase I (B. efferthii-derived). The effects of freeze/thaws, and the relative expression of different miR-29 isoforms were also assessed using RT-PCR.

Key findings: Our investigations determined that heparinase 1 (recombinant F. Heparinum) and 2 (recombinant P. heparinus) at 1 or 2 h incubation efficiently neutralized heparin activity and prevented interference with the PCR. Also, a single freeze/thaw did not affect the measurement of miR-29-3p but multiple freeze/thaw cycles decreased the measureable miR levels. Finally, the -3p strand was most abundantly expressed in all three isoforms in both human and mouse plasma.

Significance: Our findings illustrate that specific conditions need to be optimized for the particular miR and the type of sample being tested.

Keywords: Biobanked samples; Heparin; Isoform; microRNA.