Serum microRNA profiling by small RNA sequencing of healthy, hypertensive or obese pediatric individuals

To examine the effect of pediatric hypertension and/or obesity in circulating microRNA levels, microRNAs in RNA extracted from serum samples were quantified by small RNA sequencing. The serum microRNA profiling was performed for four cohorts of individuals of pediatric age-group (eight individuals per cohort). The individuals were from a patient population at a university-associated children's hospital in a large metro area of northeastern USA. The cohorts were: no diagnosis of either hypertension or obesity; diagnosis of hypertension but not obesity; diagnosis of obesity but not hypertension; and, diagnosis of both hypertension and obesity. For each individual, RNA was isolated from 0.2 ml serum using three different RNA isolation kits: Norgen Biotek Plasma/Serum RNA Purification Mini Kit(product# 55000), with RNA eluted in 15 ul; Qiagen miRNeasy Serum/Plasma Kit (#217184), with RNA eluted in 15 ul; and, Ambion mirVana PARIS Kit with phenol usage (#AM1556), with RNA eluted in 100 ul. Small RNA sequencing libraries were prepared using 6 ul of each RNA sample and the Norgen Biotek Small RNA Library Prep Kit for Illumina (#63600) using 19 PCR cycles. The libraries were sequenced on Illumina HiSeq 2500 (24 libraries per lane) to obtain single-end 51 base reads.