ORB's small RNA sequencing service

Overview

Small non-coding RNAs, which are less than 200 nucleotides (nt) in length, comprise a diverse category of regulatory RNAs that includes microRNA, piwi-interacting RNA (piRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), small nuclear RNA (U-RNA or snRNA), tRNA-derived small RNA, etc.1.These RNAs impact the transcription, processing, and stability of long RNA including messenger RNA (mRNA) through a variety of mechanisms2. The diversity in small RNA classes and functions supports the need for unbiased examination of the compendium of RNA species in biological samples and changes in their expression in response to disease, experimental manipulation, and drug treatment.The second generation sequencing platform developed at Illumina Corp. can be harnessed to generate high coverage sequencing of small RNAs cost-effectively and reproducibly, allowing accurate measurements of the levels of a wide variety of small RNAs in a single experiment3. A detailed understanding of the mechanism of microRNA binding to mRNA combined with advancement in the ability to immunoprecipitate microRNA-mRNA complexes has recently enabled identification of potential targets of microRNA regulation by sequencing approaches4

Small RNA sequencing has become a routine component of biomarker discovery initiatives as the dysregulation of microRNA and other small RNAs are implicated in many diseases5. Specifically, circulating small RNA have confirmed associations with cancer, myocardial injuries, hepatic diseases, and neurodegenerative diseases6-9

Small RNA Sequencing at ORB

ORB provides complete services for characterization and measurement of the levels of a wide variety of small RNA classes by sequencing on Illumina instruments. The core service includes evaluation of the quality of submitted RNA, preparation of libraries using the TriLink Biotechnology's CleanTag or New England Biolab's NEBNext Small RNA Sequencing Library Prep kits and sequencing on the Illumina 2500 or NextSeq 500 instruments. The advantages of ORB's small RNA sequencing service are customization for each client, the capability to handle a wide variety of challenging samples, and the company's expertise in data processing, analysis, and interpretation of results.

For each project, ORB scientist prepare a written sample processing and data analysis plan that is tailored to the research objectives of the client. Library preparation, sequencing, and analysis protocols can be tailored to target several different classes of small RNA including mature microRNA, pri-miRNA, piRNA, snoRNA, scaRNA, snRNA, vault RNA, and others.

If desired ORB can provide RNA extraction from a wide range of small-RNA rich samples such as fresh-frozen tissue, FFPE tissue sections, whole blood, cell-free biofluids, exosomes, and flow-sorted and cultured cells. ORB also accepts purified total RNA submitted by clients that has been prepared by a variety of RNA isolation methods. Additional examples of sample types which are suitable for small RNA sequencing can be found below in the sample submission instructions section. Because ORB has optimized RNA handling and library preparation methods, the company can accept samples containing as little as twenty  (20) nanograms of cellular RNA or approximately 1 nanogram from sources enriched in small RNA, such as exosomes and cell-free biofluids. This minimal requirement facilitates research on rare samples such as laser captured cells, flow sorted cells, micro-needle aspirates, micro-dissected tissue and embryonic tissue.

Depending on the source and expected mass,  ORB evaluates the quantity and integrity of isolated or directly submitted RNA by a combination of one or more techniques including UV spectrophotometry, fluorometry, denaturing gel electrophoresis, capillary electrophoresis by Bioanalyzer, and real-time PCR. Although the small RNA sequencing process is very tolerant of RNA degradation, in rare cases where the RNA quality or mass is not adequate ORB encourages its clients to submit replacement samples with no additional charge applied for the extra sample handling and quality control testing.
 
NEBNext Small RNA Library Preparation Kit - Percentage of Reads Mapped by Category
Fig 1. NEBNext Small RNA Library Preparation Kit - Percentage of Reads Mapped by Category

In order to target specific small RNA classes and keeping in mind the intended applications for the RNA beyond small RNA sequencing, ORB typically tailors its protocols for pre-fractionation of RNA and gel excision of library molecules in order to target RNA molecules of a narrow size range consistent with the target RNA classes. Figure 1 shows the recovery of sequence reads as a percentage of adapter-trimmed reads from a typical experiment designed to target mature microRNAs from human tissue; in this case gel excision was designed to recover libraries containing cDNA copies of small RNAs ranging from 17-35 nucleotides in length. Pri-miRNA is covered because the mature microRNA sequences are found within the larger pri-miRNA sequences. Fragments of tRNA and rRNA often make up a portion of reads in small RNA libraries. A percentage of reads map to that cDNA database because larger transcripts often contain microRNA loci.


ORB’s fully automated and optimized small RNA sequence analysis pipeline facilitates processing of data following the completion of a sequencing run. The process begins by filtering sequence reads for quality, trimming adapter sequences, and aligning trimmed reads to the genome and mRNA sequences. Sequences that align to the genome are further aligned to a variety of small RNA databases as well as the Ensembl transcript database, and read counts are extracted. Analysis of the read counts with flexible statistical analysis and graphing software enables preparation of full reports for small RNA biotypes of interest. ORB can also provide novel microRNA prediction and microRNA-target pathway analysis.  Please see ORB's small RNA sequencing bioinformatics page to view more information about data processing and analysis options as well as a sample project report.

In addition to supporting pure research application, ORB offers analysis services tailored to biomarker discovery programs including predictive modeling in order to identify sets of microRNAs who's expression level changes are predictive of patient prognosis, likelihood of positive response to a drug, or correlative of a desired or adverse response to a drug.  More information about potential applications of ORB's small RNA sequencing services can be found in the Related Services section below.

Highlights of ORB's small RNA sequencing services

  • Customization in order to target precise size ranges of small RNA
  • Input requirements a low as 20 nanograms cellular RNA or 0.225 ml of biofluid
  • Customizable bioinformatic analysis to focus on RNA classes of interest to client
  • NextSeq v2 chemisty
  • HiSeq 2500 v4 sequencing
  • Plate- and microfluidic array-based real-time PCR assays for validation of experimental results.
  • Novel MicroRNA prediction report
  • Analysis of other small RNAs like piRNA, snoRNA, & snRNA
  • MicroRNA target prediction
  • Full statistical analysis & alignments
  • Custom analysis reports on customer request

Processing Options
Preparation of Sequencing Libraries

  • Choice of TriLink CleanTag, or NEBNext library construction methods.
  • Adapter segregation technology for increased sensitivity.
  • Two separate gel isolations for low input projects produce high-purity libraries.
  • Flexibility to focus sequencing on multiple classes of small RNA including miRNA, piRNA, tRNA, and snoRNA.


Illumina Sequencing

  • NextSeq500 (Rapid)
  • HiSeq2500 (Rapid or High-Output mode)
  • Selection of depth, pairing and length include
  • 50 nt, 75 nt (NextSeq500) and 100 nt
  • Single and paired-end

Data Processing, Analysis, and Delivery

  • Unique sequence list with counts for all samples and list of known annotations
  • Ensembl transcript database mapping enables investigation of 40 additional RNA biotypes
  • Novel microRNA prediction based on peer-reviewed MirPara 6.2 software
  • Analysis of read counts based on either perfect match or single-mismatch alignment filtering.

Please view our small RNA sequencing data analysis page for more information about ORB's standard analysis pipeline and additional capabilities.

Figure 2.  Several steps in ORB's small RNA sequencing service facilitate customization, such as the gel fractionation which concentrates the query on a decided size range of small RNA as well as bioinformatic analysis to focus on RNA classes of interest to client.

Sample Submission Instructions

ORB's preparative services include the isolation of low-molecular weight RNA from most sample types. ORB scientists are continually optimizing RNA extraction methods for unique sample types; contact us to discuss processing options for samples not listed below.

  • FFPE tissue
  • Tissues
  • Immunoprecipitated RNA
  • HITS-CLIP or PAR-CLIP RNA
  • Cells
  • Flow sorted cells
  • LCM cells
  • Exosomes
  • Whole Blood
  • Serum
  • Plasma
  • Saliva 
  • Urine
  • Cerebrospinal Fluid
  • Lymphatic Fluid
  • Ascites Fluid
  • Synovial Fluid
  • Milk
  • Lavage

ORB also provides guidance for upstream sample collection steps, enabling clients to maximize the information captured from rare and precious samples.  Contact us for more information as you prepare for your small RNA sequencing study.

Table I. Input requirements and sample submission documents for small RNA sequencing.

Sample Type Fraction Required Input Preparation Instructions Sample Submission Checklist
Cell-free biofluid        Total 0.225 ml Biofluid - total fraction small RNA
Cell-free biofluid           Microvesicle/ 
exosome
1.0 ml
  •  Same as cell-free biofluid - total fraction 
Biofluid - exosome  small RNA
Whole Blood Total 1 PAXgene tube PAXgene - small RNA
Tissue Total 1.5 mg Tissue - small RNA
Cells Total 5x10^4 Cells - small RNA
RNA Total 20 ng RNA - small RNA sequencing

ORB offers a lower cost standard RNA input small RNA sequencing option for clients able to submit greater than 100 nanograms of total RNA or tissue or cells that yield that quantity of RNA.

When shipping, enclose a completed sample submission checklist in a separate dry compartment with the sample shipment. Also send a digital file version of the sample submission checklist to array@oceanridgebio.com and provide notification of the sample shipment to by calling 754-600-5128 on the day samples are shipped.

Example studies using ORB's small RNA Sequencing Service

Reproductive Science / Infectious Disease

Vojtech_et_al_seminal-exosomes-small-RNA-immunosuppressive-agents
Figure 3. Vojtech et. al demonstrated that exosomes derived from seminal plasma contain exosomal small RNAs associated with immunomodulatory features.

While seminal fluid content is largely comprised of organic and inorganic components including spermatozoa, fructose, citric acid, and lipids, seminal plasma constituents also include microparticles commonly categorized as exosomes which harbor immunosuppressive nucleic acids10,11. A collaboration between the University of Washington and the Fred Hutchinson Cancer Research Center recruited ORB scientists to produce cDNA libraries from small RNA isolated from seminal exosomes and to perform small RNA sequencing using a HiSeq 2000 system.  This comprehensive investigation of RNA less than 100 nt from seminal plasma revealed that over 50% of extracellular RNA content is contained within exosomes12. Moreover, detectable small RNAs included known and novel microRNAs as well as tRNAs, YRNAs and protein coding mRNA fragments which are associated with modulation of the immune systems. Findings from this study indicate that seminal exosomes contain regulatory small RNAs which influence the immunophysiology of the genital tract muscoa and increase rate of transmission of infectious diseases in the recipient.  

Dermatology

A-schematic-illustration-of-the-initiation-of-acute-atopic-dermatitis-AD
Figure 4. Dhingra, et al. illustrate the mechanistic pathway initiating the development of atopic dermatitis which includes marked immune activation and the recruitment of inflammatory cells.

Although pediatric atopic dermatitis (AD) is the most common inflammatory cutaneous disease in children, the pathogenesis and effective treatment strategies remain ill-defined13.  While AD is characterized by distinct external features such as a defective permability of the stratum corneum and antimicrobial function14,15, abnormal filaggrin expression and irregularities in the adaptive immune system are also established lead to the pruritic, inflammatory dermatosis which correlates with AD16,17. Another perspective on the etiology of AD is the hygiene hypothesis which suggests that limited exposure to microoganisms in infancy and early childhood can lead to imbalances in the internal and external microbiome and ultimately result in conditions such as AD18.  In fact, recent advances in sequencing technologies have enabled the examination of microorganisms from a wide range of biospecimens, including skin biopsies, and now the dysregulation of the skin immune response in AD is established to be associated with the cutaneous microbiota composition19,20.

The prevention and primary treatment of pediatric AD through probiotic supplementation during pregnancy and infancy is a relatively cost-effective strategy that is of great interest to the research community21.  To this end, The Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) trial in Norway investigated the effects of maternal perinatal probiotic supplementation on the cumulative incidence of pediatric AD22.  In this study, over 400 pregnant women were randomized to receive sterile milk or milk supplemented with Lactobacillus rhamnosus GGBifidobacterium animalis subsp. lactis Bb-12, and Lactobacillus acidophilus La-5 on a daily basis from 36 weeks gestation until 3 months postnatal.  The overall findings of the ProPACT trial demonstrated that the administration of probiotics resulted in an approximate 40% reduction in the incidence rate of AD development in offspring at 2 years of age.  

In a subsequent study aimed at the elucidation of the mechanism of action underlying the protective role that maternal probiotic supplementation plays in pediatric AD prevention, a team from the Norwegian University of Science and Technology utilized ORB's sequencing service to examine the small RNA content in breast milk obtained from the ProPACT trial23  RNA fragments of 11-28 nt, which derived from extracellular vesicles obtained from milk samples collected at 3 months postpartum, were analyzed using a HiSeq 2000 instrument to generate an average of 34.7 million single-end reads per sample, and detectable sequences were aligned to mature microRNA, rRNA, tRNA, and other small RNA databases.  The results of this study revealed almost 3,500 unique potential gene targets for the 20 most abundant breast milk microRNAs.  The predicted targets concentrate on metabolic processing of nitrogen compounds, embryogenesis, angiogenesis, catabolism, and cellular migration. Also, a correlative effective was demonstrated between maternal probiotic supplementation and the up- or down-regulation of let-7d-3p, miR-574-3p, miR-340-5p and miR-218-5p, and individual microRNA targets showed associations to AD or allergy related diseases, such as the suppressor of cytokine signalling 3,  T-cell differentiation and activation, and transforming growth factor β.

Related Services

Noninvasive sampling is an ideal method to assay for screening, diagnostic, or prognostic biomarkers, and to support the examination of small RNAs in biofluid samples, ORB scientists developed protocols for ultra-low input requirements from many fluids. To support cutting-edge wet lab small RNA sequencing techniques, ORB computational biologists conduct target microRNA-gene pair prediction, enriched pathway analysis of genes targeted by microRNAs, or employ statistical models to predict the best candidate biomarkers. Reproducibility is a requisite for all biomarker and companion diagnostic discovery studies, and to this end, ORB offers validation assays using real-time qPCR assays to confirm the utility of a marker set.  Confirmation studies can be certified GLP for clients' preparation of subsequent FDA or patent applications, and for every project, ORB provides supporting service to generate figures for manuscripts, posters, or presentations. Follow the links below for details about the services which complement ORB's small RNA sequencing service.


Contact us by email or phone to discuss how ORB's small RNA sequencing service can further your research!

 

References

  1. Kim, V. N. (2005). Small RNAs: classification, biogenesis, and function. Mol cells19(1), 1-15.
  2. Zhang, C. (2009). Novel functions for small RNA molecules. Current opinion in molecular therapeutics11(6), 641.
  3. Wang, Z., Gerstein, M., & Snyder, M. (2009). RNA-Seq: a revolutionary tool for transcriptomics. Nature reviews genetics10(1), 57-63.
  4. Bracken, C. P., Scott, H. S., & Goodall, G. J. (2016). A network-biology perspective of microRNA function and dysfunction in cancer. Nature Reviews Genetics, 17(12), 719-732.
  5. Williams, Z., Ben-Dov, I. Z., Elias, R., Mihailovic, A., Brown, M., Rosenwaks, Z., & Tuschl, T. (2013). Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations. Proceedings of the National Academy of Sciences110(11), 4255-4260.
  6. Chen, X., Ba, Y., Ma, L., Cai, X., Yin, Y., Wang, K., & Li, Q. (2008). Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell research18(10), 997-1006.
  7. Adachi, T., Nakanishi, M., Otsuka, Y., Nishimura, K., Hirokawa, G., Goto, Y., & Iwai, N. (2010). Plasma microRNA 499 as a biomarker of acute myocardial infarction. Clinical chemistry56(7), 1183-1185.
  8. Zhang, Y., Jia, Y., Zheng, R., Guo, Y., Wang, Y., Guo, H., & Sun, S. (2010). Plasma microRNA-122 as a biomarker for viral-, alcohol-, and chemical-related hepatic diseases. Clinical chemistry56(12), 1830-1838.
  9. Li, X., Khanna, A., Li, N., & Wang, E. (2011). Circulatory miR-34a as an RNA-based, noninvasive biomarker for brain aging. Aging (Albany NY)3(10), 985-1002.
  10. Owen, D. H., & Katz, D. F. (2005). A review of the physical and chemical properties of human semen and the formulation of a semen simulant. Journal of andrology26(4), 459-469.
  11. Madison, M. N., Roller, R. J., & Okeoma, C. M. (2014). Human semen contains exosomes with potent anti-HIV-1 activity. Retrovirology11(1), 102.
  12. Vojtech, L., Woo, S., Hughes, S., Levy, C., Ballweber, L., Sauteraud, R. P., & Hladik, F. (2014). Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic acids research, gku347.
  13. Boguniewicz, M., & Leung, D. Y. (2011). Atopic dermatitis: a disease of altered skin barrier and immune dysregulation. Immunological reviews242(1), 233-246.
  14. Leung, D. Y., Boguniewicz, M., Howell, M. D., Nomura, I., & Hamid, Q. A. (2004). New insights into atopic dermatitis. The Journal of clinical investigation113(5), 651-657.
  15. Leung, D. Y. (2000). Atopic dermatitis: new insights and opportunities for therapeutic intervention. Journal of Allergy and Clinical Immunology105(5), 860-876.
  16. Proksch, E., Fölster-Holst, R., & Jensen, J. M. (2006). Skin barrier function, epidermal proliferation and differentiation in eczema. Journal of dermatological science43(3), 159-169.
  17. Baker, B. S. (2006). The role of microorganisms in atopic dermatitis. Clinical & Experimental Immunology144(1), 1-9.
  18. Kalliomäki, M., Salminen, S., Arvilommi, H., Kero, P., Koskinen, P., & Isolauri, E. (2001). Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. The Lancet357(9262), 1076-1079.
  19. Grice, E. A., Kong, H. H., Conlan, S., Deming, C. B., Davis, J., Young, A. C., & Turner, M. L. (2009). Topographical and temporal diversity of the human skin microbiome. science324(5931), 1190-1192.
  20. Kong, H. H., Oh, J., Deming, C., Conlan, S., Grice, E. A., Beatson, M. A., & Turner, M. L. (2012). Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome research22(5), 850-859.
  21. Lee, J., Seto, D., & Bielory, L. (2008). Meta-analysis of clinical trials of probiotics for prevention and treatment of pediatric atopic dermatitis. Journal of Allergy and Clinical Immunology121(1), 116-121.
  22. Dotterud, C. K., Storrø, O., Johnsen, R., & Øien, T. (2010). Probiotics in pregnant women to prevent allergic disease: a randomized, double‐blind trial. British Journal of Dermatology163(3), 616-623.
  23. Simpson, M. R., Brede, G., Johansen, J., Johnsen, R., Storrø, O., Sætrom, P., & Øien, T. (2015). Human Breast Milk miRNA, Maternal Probiotic Supplementation and Atopic Dermatitis in Offspring. PloS one10(12), e0143496.
  24. Dhingra, N., & Guttman-Yassky, E. (2014). A possible role for IL-17A in establishing Th2 inflammation in murine models of atopic dermatitis. Journal of Investigative Dermatology134(8), 2071-2074.