Profiling of RNA and Proteins in Biofluids
Biological fluids represent an ideal source of material for biomarker development projects utilizing genomics-based assays. Extracellular RNA, DNA, and some proteins are stably maintained in cell-free biofluid samples even in cases where sample handling is not optimal. Additionally, biofluids derived from the blood and/or associated with respiration, digestion, or excretion are able to be collected routinely with minimal disruption to the health or daily schedule of a patient. Circulating biomolecules can be associated with the severity and progression of a disease as well as the efficacy of a treatment1-3; thus suitable marker sets may be discovered which can enable the development of predictive or prognostic biomarkers or companion diagnostics that can reduce the time from discovery to market approval for a therapeutic agent. Follow the links below for more background information about these applications.

Biomarker Discovery
Biomarker Discovery
Companion Diagnostics
Companion Diagnostics

ORB provides end-to-end customizable solutions to examine the levels of macromolecules in biofluids utilizing several platforms including sequencing of long and small RNA, multiplex immunoassays, as well as targeted sequencing of oncogenes and methylation sequencing of circulating cell-free DNA (cfDNA). ORB has extensive experience working with the circulatory system-based biofluids such as whole blood, serum, and plasma, but has also worked with less accessible biofluids such as rhinopharyngeal lavage, lymph, cerebrospinal, amniotic, synovial and ascites fluids, as well as dilute excreted fluids including sweat, and saliva. All ORB biofluid profiling services are optimized to accommodate samples with limited volumes. In order to overcome the increased experimental variability associated with analyzing RNA from whole biofluids, ORB has adopted commercial protocols for pre-isolation of microvesicles from cell-free biofluids allowing the profiling of primarily exosome-specific RNA molecules. Because it has been established that cell free DNA (cfDNA) levels are elevated in association with specific diseased states including cancer, and that oncogene mutational status can be monitored in cfDNA4, ORB scientists have developed an oncogene sequencing service specifically tailored to cfDNA. Investigation of global methylation patterns of cfDNA within liquid biopsies is also available. ORB provides bioinformatic services to enable identification and selection of predictive sets of biomarkers using the data collected from global RNA, DNA and protein profiling studies mentioned above. Additionally, post project support is available for all studies that ORB conducts in order to facilitate clients' preparation of FDA and patent applications as well as the generation of manuscripts and posters.

Available Services

Sequencing of long RNA in exosomes ORB offers characterization of exosomal mRNA in cell-free biofluid samples such as plasma and serum using Illumina sequencing technology starting from a minimum of 1 ml of biofluid. ORB is also able to provide non-exosome-specific sequencing of long RNA from blood samples collected in PaxGene tubes. ORB’s long RNA sequencing data analysis services define sample composition such that in addition to protein-coding RNA, many other RNA biotypes can be detected and accurately classified including, processed pseudogenes, antisense, ribsomal RNA, small nuclear RNA, and mitochondrial RNA fragments.

Sequencing of small RNA in total biofluid and exosomes ORB’s small RNA sequencing services are optimized to interrogate over 6 types of small RNAs within a variety of biofluid samples including dilute samples such as saliva. Additionally, because exosomes are well-known to show enrichment of small RNA species, ORB has developed protocols to accurately assess the small RNA content of exosomes within biofluids.

Microarray profiling of microRNA from total biofluid and exosomes Examine levels of mature microRNA in total biofluid or circulating microvesicles/ exosomes. Utilizing a proprietary microRNA microarray along with optimized RNA extraction, labeling, and hybridization processes, ORB is able to monitor global microRNA expression using small volumes of biofluids at an affordable price. Client's have utilized this microarray service to identify biomarkers associated with active inflammatory bowel disease and early-stage pancreatic cancer (see disease areas areas below).

Multiplex immunoassay with biofluids A Luminex-based assay service is provided that allows for parallel analysis of up to 60 analytes using as little as 60 microliters of biofluid. ORB provides a complete service including consultation, assay selection and configuration, pilot assay development, screening, and data analysis. With a focus on customer service and reliability ORB can help you conserve precious clinical biofluid samples by minimizing usage and freeze-thaw cycles. Assay panels are available for several disease areas, such as oncology, neuroscience, cardiovascular disease, endocrinology, kidney toxicity, liver and vascular injury. GLP service is available.

Oncopanels Service includes isolation of cell-free DNA from small volumes of biofluid, preparation of amplicon libraries, and sequencing on the NextSeq 500 instrument. Amplicon panels from SwiftBio and Illumina enable high coverage sequencing of the coding regions of up to 94 oncology-related genes. Reports are prepared containing lists of known and novel variants, with available disease indication and prognosis, allelic frequencies, and confidence scores for each patient.

cfDNA Bisulfite Sequencing Recent advances in library preparation have enabled sequencing-based characterization of DNA methylation patterns from circulating cell-free DNA (cfDNA). ORB provides a complete service including DNA extraction, library preparation, bisulfite sequencing, and analysis. Combine bisulfite sequencing with any of the services mentioned above to leverage the power of a multi-modal approach to biomarker discovery. 

Real-Time qPCR Assays Validate biofluid profiling results from microarrays or sequencing using precise and reliable quantification of the levels of specific mRNAs, microRNAs, and other RNA types using ABI Taqman quantitative PCR. ORB can provide GLP ABI Taqman projects upon request to advance your therapeutics and/or companion diagnostics program.

Applications

Biomarker Discovery

Circulating biomarkers are now established to be relatively stable in most biofluids, and, unsurprisingly, these markers have been shown to contribute to many pathological processes5. Advances in highly sensitive techniques such as massively parallel sequencing and multiplex protein profiling have facilitated the discovery of novel mRNA, microRNA, long non-coding RNA, and protein biomarkers within biofluid samples where starting materials can be limited and biomolecules of interest are of low abundance6-10.

ORB routinely processes biofluid samples to quantify established biomarkers or to discover novel markers associated with the presence or progression of a pathology or the efficacy of a treatment. ORB’s biomarker discovery services enable the identification of markers which can be utilized to stratify patients, distinguish non-responders, and provide early detection screening. The identification and development of early detection markers for diseases with aggressive progression and where patients remain without symptoms until late-stage development is important to improve patients survival rates. Additionally, circulating biomarkers provide clinicians a minimally invasive tool to assess a patients’ health status or monitor the effects of a therapeutic in an outpatient setting which reduces costs and the introduction of noncosimal infections which are commonly associated with procedures to obtain tissue biopsies.

ORB can assist companies with development of clinical assays through the discovery of biomarkers, validation, patent submission, and FDA applications for companion diagnostics. For additional information regarding how ORB’s biofluid profiling services can support your drug discovery and development program contact us.

Disease Areas

Biofluid profiling is particularly germane for preventative screening and with diseases where patients appear asymptomatic in the early stages such as pancreatic cancer. One investigation of early-detection biomarkers for stage II pancreatic ductal adenocarcinoma from plasma samples utilized ORB’s proprietary microRNA microarray and validation services with ABI Taqman assays. From this work, three blood-based circulating microRNAs targets (miR-642b-3p, miR-885-5p, and miR-22-3p) were shown to be significantly up-regulated in cancer patients. (Ganepola GA et al., 2014).

In cases of chronic diseases that cycle between episodes of activity and remission, relatively non-invasive biofluid analysis facilitates routine testing to monitor the disease state and potentially provide advance notification of an active period. An example utilizing ORB's biofluid analysis to develop such markers was published in 2015 by Dr. Geoff Krissansen and colleagues at the University of Auckland (Krissansen GW et al., 2015). Using a microRNA microarray for discovery and real-time qPCR for confirmation, ORB collected evidence that miR-595 and miR-1246 expression levels were associated with active versus inactive disease states for patients with ulcerative colitis and Crohn’s disease (Krissansen GW et al., 2015). In a separate study, ORB’s analysis of TGF-B and IL-10 with plasma samples using ELISA assays was employed to examine potential immunoregulatory signatures associated with inflammatory bowel disease (Gurram B et al., 2016).

The development of biofluid-based biomarkers for non-invasive prenatal testing in order to predict potential complications of pregnancy is of great interest. In particular, changes in the levels of specific circulating microRNAs may be predictive of an increased risk of developing preeclampsia during the later stages of pregnancy.11 To this end, ORB’s microRNA microarray analysis services were utilized to examine maternal plasma samples collected in early-mid pregnanacy for circulating markers for associations with pre-pregnancy body mass index. The results indicated over 25 microRNAs were differentially expressed and showed a positive association with maternal pre-pregnancy overweight and obese status (Enquobahrie, et al., 2016).

Cancer, both hematological and solid tumors, has been shown to possess mutations which are unique to spatial and temporal sampling12,13, therefore analysis of circulating biomarkers in biofluid samples is considered to provide a potentially better representation of the disease status. Common diseases areas in which biofluid profiling holds great utility are listed below:

  • Oncology
  • Cardiovascular
  • Metabolic Diseases
  • Infectious Diseases
  • Endocrinology
  • Neurological Diseases/Disorder
  • Respiratory
  • Ocular
  • Inflammatory Bowel Disease
  • Nephrology
Biofluid profiling enables improved patient management strategies using minimally invasive sample collection strategies.

 


Obtain detailed information on specific biofluid-related services by following the links below, use our contact form to reach out to us, or phone us today at 754-600-5128 to start planning your biofluid profiling study. We look forward to assisting you!

 

 
References
  1. Quinn, J. F., Patel, T., Wong, D., Das, S., Freedman, J. E., Laurent, L. C., & Spetzler, R. (2015). Extracellular RNAs: development as biomarkers of human disease. Journal of extracellular vesicles4.
  2. Hassanein, M., Callison, J. C., Callaway-Lane, C., Aldrich, M. C., Grogan, E. L., & Massion, P. P. (2012). The state of molecular biomarkers for the early detection of lung cancer. Cancer prevention research, canprevres-0441.
  3. Rennert, R. C., Hochberg, F. H., & Carter, B. S. (2016). ExRNA in Biofluids as Biomarkers for Brain Tumors. Cellular and molecular neurobiology36(3), 353-360.
  4. Wielscher, M., Vierlinger, K., Kegler, U., Ziesche, R., Gsur, A., & Weinhäusel, A. (2015). Diagnostic performance of plasma DNA methylation profiles in lung cancer, pulmonary fibrosis and COPD. EBioMedicine2(8), 929-936.
  5. 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.
  6. 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.
  7. Akat, K. M., Moore-McGriff, D. V., Morozov, P., Brown, M., Gogakos, T., Da Rosa, J. C., & Totary-Jain, H. (2014). Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers. Proceedings of the National Academy of Sciences111(30), 11151-11156.
  8. Kiko, T., Nakagawa, K., Tsuduki, T., Furukawa, K., Arai, H., & Miyazawa, T. (2014). MicroRNAs in plasma and cerebrospinal fluid as potential markers for Alzheimer's disease. Journal of Alzheimer's Disease39(2), 253-259.
  9. Guo, W., Wang, Q., Zhan, Y., Chen, X., Yu, Q., Zhang, J., & Zhu, L. (2016). Transcriptome sequencing uncovers a three–long noncoding RNA signature in predicting breast cancer survival. Scientific Reports6.
  10. Domenici, E., Willé, D. R., Tozzi, F., Prokopenko, I., Miller, S., McKeown, A., & Holsboer, F. (2010). Plasma protein biomarkers for depression and schizophrenia by multi analyte profiling of case-control collections. PLoS one5(2), e9166.
  11. Ura, B., Feriotto, G., Monasta, L., Bilel, S., Zweyer, M., & Celeghini, C. (2014). Potential role of circulating microRNAs as early markers of preeclampsia. Taiwanese Journal of Obstetrics and Gynecology53(2), 232-234.
  12. Hiley, C., de Bruin, E. C., McGranahan, N., & Swanton, C. (2014). Deciphering intratumor heterogeneity and temporal acquisition of driver events to refine precision medicine. Genome biology15(8), 453.
  13. Sottoriva, A., Spiteri, I., Piccirillo, S. G., Touloumis, A., Collins, V. P., Marioni, J. C., & Tavaré, S. (2013). Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proceedings of the National Academy of Sciences110(10), 4009-4014.