Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

Abstract

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. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina's TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.

High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

“…For the primary data sets, plasma RNAs were extracted by using TRIzol LS Reagent (Thermo Fisher Scientific) followed by a Direct-zol RNA MiniPrep Kit (Zymo Research), as described in Materials and Methods. This method, which we refer to as the Direct-zol method, typically recovered 2-8 ng of nucleic acids per milliliter plasma, comparable to yields in previous studies (Burgos et al 2013;Williams et al 2013;Spornraft et al 2014). The plasma RNA samples were analyzed by RNA-seq with no further treatment (NT), after enzymatic treatment to remove 3 ′ phosphates (-3 ′ P), which block TGIRT template-switching (Mohr et al 2013), or after on-column DNase I digestion (OCD) under conditions that completely digest 10 ng of a mixture of a 74-nt ssDNA and a 275-nt dsDNA PCR product (Supplemental Fig.…”

“…A recent exciting application of RNA-seq is the analysis of extracellular RNAs present in plasma and other bodily fluids (Mitchell et al 2008;Burgos et al 2013;Huang et al 2013;Williams et al 2013;Koh et al 2014). Such extracellular RNAs are potential biomarkers for human diseases and may be involved in intercellular communication (Valadi et al 2007;Zernecke et al 2009;Fabbri et al 2012;Grasedieck et al 2013).…”

Survey of 800+ data sets from human tissue and body fluid reveals xenomiRs are likely artifacts

“…The standard Illumina protocol for sRNA-seq library preparation requires relatively large amounts of RNA (>1 μg of total RNA). This is easily obtained with tissue samples, but it can be difficult to extract these amounts of RNA from body fluids (Burgos et al 2013). Therefore, sequencing of small RNAs from body fluids often use lower amounts of input material, in some cases down to 10 ng or 100 µL of input fluids for the RNA extraction (Wang et al 2012(Wang et al , 2013, and this can be compensated through additional cycles of PCR amplification (Wang et al 2012).…”

Divergent MicroRNA Targetomes of Closely Related Circulating Strains of a Polyomavirus

“…The small RNAs from strain 776 infection was subjected to SOLiD sequencing as previously described (40,41). The small RNAs from RI257-MIR virus infection were subjected to Illumina sequencing as described previously (48).…”