Taqman mirna assay primers

RT-qPCR was performed to determine the novel miRNA expression levels in an independent sample of 881 normotensive, screen-detected hypertensive and known hypertensive male and female participants, as determined by next generation sequencing. Briefly, using the TaqMan Advanced miRNA cDNA synthesis kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA), 2 µl of total RNA was converted into cDNA through sequential conduction of poly-A tailing, adaptor ligation, reverse transcription and miR-Amp steps as per the manufacturer’s recommendation. The miRNA expression levels were then determined using TaqMan miRNA Assay primers (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA) on the QuantStudio 7 Flex real-time PCR instrument (Life Technologies, Carlsbad, CA, USA). In order to determine relative miRNA expression in each sample, the 2^−ΔCt method was used whilst the 2^−ΔΔCt method was used to compute fold change differences in miRNA expression between the study groups using miR-16-5p (ThermoFisher Scientific, Waltham, MA, USA) as the endogenous control [19 (link)].

Gene expression was measured using quantitative real-time PCR (qPCR). All mRNA primers are described in S4 Table. Gene expression was normalized to the expression of the internal control gene ribosomal protein L7a (L7a). For miRNA expression, qPCR was performed using TaqMan miRNA Assay primers that were specific to miR-105 or miR-767 (Applied Biosystems) and miRNA were quantified via the ΔΔCT method. A TaqMan miRNA Control Assay for U6 snRNA was included for normalization. miRNA primers can be found in S5 Table. A detailed PCR protocol can be found in the S1 Supplementary Methods.

In order for miRNA expression to be determined using RT-qPCR, the extracted RNA had to be converted to cDNA first and this conversion was done using the TaqMan Advanced miRNA cDNA synthesis kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, United States) as per the manufacturer’s recommendations. In summary, by sequentially conducting poly-A tailing, adaptor ligation, reverse transcription and miR-Amp steps, we converted 2 μl of total RNA into cDNA, which was the starting material in the succeeding PCR step used to determine miRNA expression levels. This PCR analysis was conducted on the QuantStudio 7 Flex real-time PCR instrument (Life Technologies, Carlsbad, CA, United States) using TaqMan miRNA Assay primers (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, United States). The determination of relative miRNA expression in a sample was done using the 2^–ΔCt, whilst fold change differences in miRNA expression between the study groups were computed with the use of the 2^–ΔΔCt method. In order to normalize miRNA quantification, miR-16-5p (ThermoFisher Scientific, Waltham, MA, United States) was used as the endogenous control (Livak and Schmittgen, 2001 (link)).