Library Preparation for Small RNA Transcriptome Sequencing in Patients Affected by Cerebral Cavernous Malformations

Abstract

Small RNA sequencing by Illumina’s Next Generation technology has revolutionized the transcriptome analysis by facilitating massive parallel sequencing of RNA molecules at low cost. Illumina’s Next Generation RNA sequencing is ideal for profiling small RNA (microRNAs, snoRNAs, and piRNAs) libraries in the identification of novel biomarkers for better clinical diagnosis. This method offers significant advantages when compared to microarray analysis with the ability to identify novel transcripts, higher sensitivity, specificity, and detection of rare and low-abundance transcripts. Small RNAs, including microRNAs and snoRNAs, belong to the class of small non-coding RNAs with 50–200 nucleotides in length and are involved in post-transcriptional regulation of gene expression. Executing Illumina’s Next Generation Sequencing technology, we have recently deciphered microRNAs and snoRNAs expressed in cerebral cavernous malformations (CCMs). Small RNA library preparation is a prerequisite step prior to RNA sequencing for the identification of microRNAs and snoRNAs. Here, we describe stepwise small RNA library preparation starting from total RNA isolated from CCMs patient until library validation using the Illumina^® TruSeq^® Small RNA Sample preparation kit. We believe this method will shed light into the functional identification of other novel small non-coding RNAs in CCMs that awaits discovery.