Quantification of miRNA by qRT-PCR

The miRNAs were quantified by qRT-PCR using a Human TaqMan MicroRNA Assay Kit (Applied Biosystems, Foster City, CA). The reverse transcription reaction was performed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). qPCR was run on a StepOnePlus PCR system (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software v2.0 (Applied Biosystems).

As previously reported [13], we used an approach for data normalization based on spiking the samples with a synthetic RNA oligonucleotide, cel-miR-39, which does not exist in the human genome. C. elegans cel-miR-39 was purchased as a custom-made RNA oligonucleotide (Qiagen, Valencia, CA). The oligo used for spiking, as a mixture of 25 fmol of oligonucleotide and water in a total volume of 5 µl, was introduced after the addition of 2X Denaturing Solution (Ambion) to the plasma sample to avoid degradation by endogenous plasma RNases. As a control for each RNA sample, cel-miR-39 was used for the TaqMan qRT-PCR assays (Applied Biosystems) as described above. We normalized the data across samples using the 2^−ΔΔCt method relative to cel-miR-39. However, the expression of miRNAs from tissue samples and cultured cells was normalized using the 2^−ΔΔCt method relative to U6 small nuclear RNA (RNU6B).