We performed qPCR according to manufacturer specifications, using two popular qPCR methods: LNA miRCuRY (Exiqon, Woburn, MA, USA) and Taqman (ThermoFisher, Waltham, MA, USA). All assays were run on an Applied Biosystems StepOne qPCR machine. For both LNA and Taqman, we performed qPCR using the commercially available probe from each company that is marketed as detecting hsa-miR-21-5p. These probes are designed to detect the canonical hsa-miR-21-5p sequence, or the 0|0 isomiR.
For each qPCR assay, we performed a standard curve experiment of serial dilutions of each isomiR, from a starting concentration of 10–13 M (100 fm) RNA stepping by a dilution factor of 10 down to a concentration of 10–18 M (1 am). This range was selected to represent the physiological range of isomiR concentrations we have previously observed in sequencing data (not shown). For each synthetic oligo dilution, qPCR was run in triplicate from a single reverse transcriptase (RT) reaction.
LNA miRCuRY qPCR and Taqman miRNA qPCR were then repeated as above, using each of these synthetic cell lines as starting material and using the qPCR probe designed for the 0|0 isomiR in each case. Triplicate qPCR was run from a single RT starting sample.
For each qPCR assay, we performed a standard curve experiment of serial dilutions of each isomiR, from a starting concentration of 10–13 M (100 fm) RNA stepping by a dilution factor of 10 down to a concentration of 10–18 M (1 am). This range was selected to represent the physiological range of isomiR concentrations we have previously observed in sequencing data (not shown). For each synthetic oligo dilution, qPCR was run in triplicate from a single reverse transcriptase (RT) reaction.
LNA miRCuRY qPCR and Taqman miRNA qPCR were then repeated as above, using each of these synthetic cell lines as starting material and using the qPCR probe designed for the 0|0 isomiR in each case. Triplicate qPCR was run from a single RT starting sample.