Specific extracellular RNAs (exRNAs) have been shown in basic and clinical studies to regulate key processes central to the pathogenesis of cardiovascular disease (CVD). A growing number of smaller human studies or studies examining a limited number of miRNAs have associated exRNA with CVD and its risk factors. In our study, we performed RNA sequencing (RNA-seq) on previously stored plasma samples from Framingham Heart Study (FHS) participants (Offspring Exam 8), including those with and without CVD, to determine if there was a multimarker predictor using exRNAs to discriminate between CVD and disease-free individuals. From these data, we plan to study a profile of approximately 600 exRNAs in almost 3000 additional participants of the FHS. Thus far, we have sequenced 20 CVD and 20 matched non-CVD plasma samples using an Ion Proton platform. Sequencing data was processed in the Genboree Sequencing pipeline and comparative analysis was performed.
Specifically, RNAs samples were isolated from plasma using a miRCURY RNA Isolation Kit –Biofluids (Exiqon, Denmark). Ion Total RNA-Seq Kit v2 (Life Technologies, USA) was used for creating libraries for sequencing. Ion Chef System and Ion PI IC 200 kits were used for template preparation, and sequencing was performed on Ion PI Chip Kit v2 BC and Ion Proton System (Life Technologies, USA).
From these data, we identified a total of 688 small RNAs above ≥5 Reads Per Million (RPM). The small RNAs were comprised of 426 miRNAs, 36 piRNAs, 24 snoRNAs and 202 tRNAs. miR-223-3p and miR451a were the top 2 most expressed miRNAs. We observed strong correlation in gene expression between the CVD and non-CVD groups. Only miR-589-3p expression was significantly changed in the CVD group compared to the non-CVD group. We have utilized these findings to develop our target exRNA list and are completing measurements by high-throughput RT-PCR in the remaining participants of the Offspring 8 cohort.