There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction.
The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. Researchers at the Translational Genomics Research Institute systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. They used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. They significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. They subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. The researchers then prepared small RNA sequencing libraries using Illumina’s TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, they found that the miRNA expression profile of CSF is substantially different from that of serum. To the authors knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.
- Burgos KL, Javaherian A, Bomprezzi R, Ghaffari L, Rhodes S, Courtright A, Tembe W, Kim S, Metpally R, Van Keuren-Jensen K. (2013) Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing. RNA 19(5), 712-22. [article]