Spearman correlation for the expression level of detected microRNAs.

<p>While different numbers of miRNAs were quantified between duplicated samples, the majority of these miRNA were commonly observed. The expression level of these commonly observed miRNA were highly correlated for both duplicated samples (r>0.9) and across samples from different subjects (r>0.74).</p

Schematic summarizing plasma sample treatments used in this study.

<p>RNA isolation was performed with or without proteinase K treatment and ribodepletion on fresh or archived samples.</p

Top 20 miRNA by read counts in duplicated control samples.

<p>Top 20 miRNA by read counts in duplicated control samples.</p

High Throughput Sequencing of Extracellular RNA from Human Plasma

<div><p>The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as ‘liquid biopsies’ for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs) using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms.</p></div

Top expressing miRNA with target genes and reported dysregulation in human disease.

<p>Top expressing miRNA with target genes and reported dysregulation in human disease.</p

Read counts in all samples analyzed.

<p>Read counts in all samples analyzed.</p

Venn diagram of mature miRNA species detected in each treatment group.

<p>Plasma samples libraries from a single health donor generated using no PK, PK treatment before GITC, PK treatment in GITC show strong concordance of miRNA species detected, with PK treatment in GITC method showing higher sensitivity than others.</p

Generation of tagged cyclin E1 knock-In mice and analyses of cyclin E1-containing protein complexes.

<p>(A and B) Targeting strategy to knock-in Flag and HA tags into <i>the cyclin E1</i> locus to generate N-terminally tagged <i>cyclin E1</i>^<i>Ntag</i> (A) and C-terminally tagged <i>cyclin E1</i>^<i>Ctag</i> alleles (B). The exons are shown as green boxes, Flag tag as a blue box, and HA tag as a red box. Start and stop codons are marked with orange and yellow arrowheads, respectively. The hygromycin resistance cassette (Hyg) with flanking loxP sequences (filled arrows) is also indicated. Restriction enzyme recognition sites: E, EcoRI; A, AflII; Sc, ScaI; N, NotI; X, XhoI; K, KpnI; S, SpeI; P, PmeI; Hp, HpaI. Note that panel (A) was shown in ref [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (C) Western blot analysis of wild-type control (Ctrl), heterozygous cyclin <i>E1</i>^{<i>+/Ntag</i>}, <i>cyclin E1</i>^{<i>+/Ctag</i>}, and <i>cyclin E1</i>^{<i>Ntag/Ctag</i>} embryonic stem cells probed with anti-cyclin E1 and -HA antibodies. Actin served as a loading control. Forth panel: cyclin E1 was immunoprecipitated with anti-Flag antibody and the immunoblots were probed with anti-Cdk2 antibody. Fifth panel: anti-Flag immunoprecipitates were used for <i>in vitro</i> kinase reactions using histone H1 as a substrate. (D) Same analyses as in (C) using spleens of homozygous knock-in mice. Lanes 1–2 in panels (C and D) were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (E) Cyclin E levels detected by western blotting in the indicated organs of 1-month-old mice and in embryonic brain (day E14.5). Actin served as a loading control. The last two lanes (Brain) were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>]. (F) Quantification of cyclin E levels in different organs, normalized against actin (from E). (G) Protein lysates from brains and testes of adult tagged cyclin E1 knock-in mice were separated by gel-filtration chromatography. Fractions containing protein complexes of the indicated molecular weights were analyzed by western blotting for cyclin E using an anti-HA antibody. (H) Cyclin E1-associated proteins were purified from the indicated organs of tagged cyclin E1 knock-in (KI) mice, or from control mice (Ctrl, ‘mock’ purifications) by sequential immunoaffinity purifications with anti-Flag and -HA antibodies, and 10% of the final eluate was resolved on PAGE gels and silver-stained. Arrows indicate bands corresponding to cyclin E1. Panels representing embryonic and adult brains were previously shown in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006429#pgen.1006429.ref008" target="_blank">8</a>].</p

Effect of miR-152 and miR-10b-5p on the expression of putative targets.

<p>(A) GSEA established that predicated targets of miR-152 and miR-10b-5p was positively correlated with MM, and negatively correlated with HD. NES: normalized enrichment score; FDR: false discovery rate. (B) The expression of predicted targets of miR-152 (DNMT1 and E2F3) and miR-10b-5p (E2F3, BTRC and MYCBP) in miR-152-, -10b-5p- or NC-transfected MM cells by real-time PCR with normalization to the reference 18S expression. ^*<i>P</i><0.05 compared with NC-transfected cells. (C) The expression of DNMT1, E2F3, BTRC and MYCBP in healthy donor and MM (GSE5900 and GSE2658).</p

Methylation of miR-152, -10b-5p and miR-34c-3p in MM cells.

<p>(A) Schematic illustration of the percentage of C + G nucleotides (CG%) and the density of CpG olinucleotides were shown for a region spanning 1000 bp upstream and 500 bp downstream of miR-152, miR-10b-5p and miR-34c-3p, respectively. Specific primers for these CpG islands were designed (arrows) and used to amplify these DNA fragments in MM cell lines. The CpG island was depicted, and each vertical bar illustrated a single CpG. (B) Representative MSP results of the three miRNAs methylation inMM1S, RPMI 8266, OPM2, U266, IM9 and H929 cell lines. M: methylated primers; U: unmehtylated primers. PC: positive control. (C) Bisulfite sequencing analysis showed relative methylation frequencies of miR-152, -10b-5p and miR-34c-3p in six MM cell lines. Eight single clones for each sample were selected and T7 primers were used for sequencing. (D) Bisulfite sequencing analysis showed methylation frequencies of miR-152 and miR-34c-3p in H929 and IM9, and miR-10b-5p in H929 and MM1S, treated with or without 5′-aza-CdR (5 µM) for 4 days. Black and white circle represented methylated and unmethylated CpG, respectively. (E) Representative sequencing results showed that the cytosine (C) residues of CpG dinucleotides were converted into thymidine (T).</p