Overview

ORB provides small RNA sequencing in order to characterize and measure levels of microRNAs, piRNAs, and other small RNAs in whole and cell-free biofluids, exosomes purified from biofluids, or whole blood samples. Whole blood contains both cellular and non-cellular fraction, and within the non-cellular fraction there are small RNAs associated with microvesicles as well as an additional class of non-microvesicle-associated small RNAs1. Other biofluids such as CSF, lavage, milk, etc., also contain both microvesicle-associated and non-associated cell-free small RNAs2,3. Exosomes, which are a special class of cell-free microvesicles ranging from 30-150 nm in size, harbor nucleic acids which can serve as diagnostic and prognostic biomarkers for many diseases4. For more information about exosomes see the main exosomal RNA profiling page.

The major advantages of small RNA sequencing over other techniques, such as microarray or real-time quantitative PCR for the characterization of circulating small RNAs, include an open-ended interrogation strategy which allows for de novo discoveries of novel small RNAs, a customizable process to interrogate small RNA molecules of selected lengths, and the opportunity to examine multiple small RNA biotypes with one assay4. Biomarker discovery and companion diagnostic programs often utilize small RNA sequencing technologies to identify circulating markers linked to the presence of a disease, prognosis of a treatment, or the efficacy of a specific therapeutic agent5.

ORB is an experienced service provider of small RNA sequencing services to examine a variety of biofluid and circulating exosome samples.  ORB scientists have optimized the small RNA sequencing process to be compatible with input requirements as low as 0.225 ml of cell-free biofluid sample for total small RNA examinations and 1.0 ml to investigate the small RNA content of microvesicles or exosomes exclusively. This workflow and all-inclusive data analysis packages enable clients to explore a range of small RNA biotypes found in biofluids and exosomes, such as rRNA, tRNA, snoRNA, mature microRNA, precursor microRNA hairpins, and piRNA. Bioinformatic services can also include the correlation of microRNA and mRNA sequencing data, and for advanced biomarker discovery studies, ORB’s microRNA target gene set enrichment analysis can be incorporated as well as predictive modeling analyses to identify genetic biomarkers that are relevant to therapeutic treatments. All ORB small RNA sequencing projects are based on a consultative approach that is guided by client-specific objectives to ensure that each assay is customized so research questions will be addressed with confidence.

Sample Submission Guidance

Typical samples accepted for biofluid and exosome small RNA sequencing include whole blood and various cell-free biofluids.  Minimal volumes of cell-free biofluid can be submitted for profiling of RNA purified from total fluid, whereas approximately a 5-fold greater amount is required for exosome small RNA profiling. Refer to Table I for information about input requirements as well as links to documents which offer guidance for sample preparation and submission.

Table I. Biofluid volume requirements and sample submission documents for biofluid and  exosomal small RNA sequencing.

Sample Type Fraction Required Volume (ml) Preparation Instructions Sample Submission Checklist
Cell-free biofluid1        Total 0.225 Biofluid - total fraction small RNA
Cell-free biofluid1           Microvesicle/ 
exosome
1.0
  •  Same as cell-free biofluid - total fraction 
Biofluid - exosome  small RNA
Whole Blood Total One PAXgene tube PAXgene - small RNA
1, ORB provides exosomal small RNA isolation services to examine a variety of cell-free biofluid samples, such as serum, plasma, bronchial lavage, urine, milk, as well as cerebrospinal, ascites, and lymphatic fluids. Contact us to discuss your specific sample type or project.


As starting material for small RNA profiling, ORB also accepts isolated exosomes or total RNA isolated from exosomes. One typical application is submission of exosomes isolated from conditioned media of cultured cells. Generally, the conditioned media from about 20 million cells should be the starting point for exosome isolation in order to yield adequate RNA for exosome small RNA sequencing. To explore the small RNA sequencing possibilities with your specific sample type or project, contact us.

ORB’s Biofluid and Exosome Small RNA Analysis Services

ORB’s biofluid exosomal small RNA profiling provides a feature-rich workflow that is adjusted to achieve researchers' specific objectives. Biofluid-based small RNA sequencing studies can begin with the isolation of exosomes from cell-free fluids and/or the extraction of low-molecular weight RNA from whole blood, cell-free biofluids and exosomes.  Quality control testing of isolated RNA includes real-time qPCR analysis for 3 microRNAs; should samples fail to pass ORB's QC assessment, the client can provide replacement samples to include in the study.  ORB scientists prepare cDNA libraries to target precise size ranges of small RNA as directed by the client, and sequencing uses the Illumina instrumentation and technology.

To support small RNA sequencing studies that interrogate biofluid samples, ORB has developed a reliable software pipeline for processing and analysis of small RNA sequencing data backed by extensive research and validation. A wide selection of customizable bioinformatics analysis services are offered to provide a comprehensive and all-inclusive package to support any data analysis requirements. State-of-the-art predictive modeling is available to facilitate biofluid-based biomarker discovery initiatives. See ORB small RNA bioinformatics page for more information about the standard analysis package, additional options, and specialty analysis capabilities. Candidate exosomal microRNA sets can be validated using real-time qPCR assays; confirmation studies can be conducted under GLP compliance to support subsequent FDA and patent applications. Find more information about specific aspects of ORB's small RNA profiling and related services by following the links below.


Small RNA Sequencing

Small RNA Bioinformatics

Real-Time qPCR

Biomarker Discovery


Demonstration - small RNA sequencing from total serum and isolated exosomes.

Exosomal Small RNA Procedures

qPCR of RNA isolated from exosomes
Figure 1: Comparison of exosome RNA isolation methods using ABI Taqman Real-Time qPCR microRNA assays.

Exosomal RNA was extracted from one (1.0) ml of serum from healthy male volunteers using three different methods: Qiagen ExoRNeasy, Norgen Serum/Plasma Exosome Purification Kit followed by ORB’s standard RNA isolation method and the Norgen Exosomal RNA Isolation Kit. MicroRNA-specific reverse transcription was conducted using the equivalent of 25 ul serum per reaction, while random primed cDNA synthesis for mRNA targets was conducted using the equivalent of 150 ul serum. Our findings indicate that the Qiagen ExoRNeasy kits provides the greatest yield of exosomal RNA as shown with the ABI Taqman RT-qPCR analysis of miRs–150, -451, and –16 with GAPDH and RPLP0 as normalization controls (see Figure 1).

The subsequent comparison of tissue, serum, and serum-Exosome RNA utilized DNA-free RNA isolated from human serum exosomes using the Qiagen ExoRNeasy kit and DNA- free total human serum RNA was isolated using ORB‘s modified Trizol process (see Table II). The TriLink Biotech Clean-Tag Small RNA KitTM with ORB modifications produced libraries containing cDNA inserts corresponding to original RNA molecules ranging from 17-35 nucleo- tides in size. Small RNA sequencing using the Illumina HiSeq-2500 generated ~35 million passed-filter single-end reads per sample.


Table II. Comparison of tissue, serum, and serum-exosome RNA with ultra-low starting input material.
 
Sample Type Input
Heart Total RNA 20 ng
Heart Total RNA 20ng
Prostate Total RNA 20 ng
Prostate Total RNA 20 ng
Serum ExoRNA - Patient #7 200 ul equiv.
Serum ExoRNA - Patient #7 200 ul equiv.
Serum ExoRNA - Patient #8 200 ul equiv.
Serum ExoRNA - Patient #8 200 ul equiv.
Serum Exosome RNA - Patient #7 1.0 ml equiv.
Serum Exosome RNA - Patient #8 1.0 ml equiv.
Water NA

Small RNA Data Analysis

Analysis with ORB’s OMAP pipeline revealed a relatively high proportion of serum exosome RNA reads mapped to the Ensembl cDNA database (see Figure 2). Reads mapping to mature miRNA varied by sample type:

• Heart and Prostate: 12-15 million
• Serum: 1.9 – 3.0 million
• Serum Exosome: 0.6 – 1.2 million

The majority of Heart and Prostate reads mapping to cDNA were potentially derived from retained introns. In contrast, serum total and exosome RNA reads mapped mainly to protein-coding transcripts – these were primarily mature microRNA sequences embedded in coding genes (see Figure 3).

Statistical Analysis of the microRNA class within the small RNA-Seq data employed one-way ANOVA and post-hoc Tukey tests for detectable mature microRNA RPM values (see Table III). A total of 498 microRNAs were selected for analysis based on detection of at least 10 mapped reads in 2 or more samples. Thirty-seven (37) microRNAs showed a significant difference between serum total and exosomal RNA (see Figure 4 for graphical representation of 16 microRNAs).

Table III: One-way ANOVA and post-hoc Tukey tests for detectable mature microRNA RPM values.
 
Statistical Analysis Results Summary for microRNA Class
Variable Name ANOVA (sample type) Tukey Exosome vs. Heart Tukey Exosome vs. Prostate Tukey Heart vs. Prostate Tukey Exosome vs. Serum Tukey Serum vs. Heart Tukey Serum vs. Prostate
Lowest P value 1.00E-17 6.42E-14 6.42E-14 6.42E-14 2.69E-07 6.42E-14 6.42E-14
Count P<0.05 333 187 229 145 64 242 266
Count FDR<0.1 341 166 227 135 37 242 267

 

"Mapping of trimmed reads to small RNA database"
Figure 2: Mapping of trimmed reads to small RNA databases (tRNA, rRNA, snoRNA, miRNA, pri-miRNA, piRNa, cDNA, and retrotransposon).

 
"Characterization of reads mapping to Ensembl cDNA database."
Figure 3: Characterization of reads mapping to Ensembl cDNA database.

 
"MicroRNAs showing difference between serum total and exosomal RNA."
Figure 4: MicroRNAs showing difference between serum total and exosomal RNA.

Interested in exosomal small RNA profiling? We would be glad to provide a complimentary consultation about how best to achieve your research goals. Contact us today!
 

References

  1. Mitchell, P. S., Parkin, R. K., Kroh, E. M., Fritz, B. R., Wyman, S. K., Pogosova-Agadjanyan, E. L., ... & Lin, D. W. (2008). Circulating microRNAs as stable blood-based markers for cancer detection. Proceedings of the National Academy of Sciences105(30), 10513-10518.
  2. Roberts, T. C., Coenen-Stass, A. M., Betts, C. A., & Wood, M. J. (2014). Detection and quantification of extracellular microRNAs in murine biofluids. Biological procedures online16(1), 5.
  3. Burgos, K. L., Javaherian, A., Bomprezzi, R., Ghaffari, L., Rhodes, S., Courtright, A., ... & Van Keuren-Jensen, K. (2013). Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing. Rna19(5), 712-722.
  4. Lin, J., Li, J., Huang, B., Liu, J., Chen, X., Chen, X. M., & Wang, X. Z. (2015). Exosomes: novel biomarkers for clinical diagnosis. The scientific world journal2015.
  5. Morin, R. D., O’Connor, M. D., Griffith, M., Kuchenbauer, F., Delaney, A., Prabhu, A. L., & Eaves, C. J. (2008). Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome research18(4), 610-621.
  6. Wu, X., Somlo, G., Yu, Y., Palomares, M. R., Li, A. X., Zhou, W., & Wang, J. (2012). De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer. Journal of translational medicine10(1), 42.