Serum, plasma and other bodily fluids contain microRNAs that can serve as biomarkers in many disease states including cancer1-2.Despite the extremely low amount of RNA found in cell-free serum samples, ORB has developed robust protocols for microarray-based profiling of microRNA in small volumes of biofluid. ORB’s proprietary multi-species microarray assay provides a cost-effective and focused solution for relative quantification of microRNAs in biofluid samples. The assay has been validated with many biofluid types and provides coverage of mature microRNAs for human, mouse, and rat. This microRNA microarray service includes extraction of RNA from biofluid samples, end-labeling of microRNAs with Genisphere’s flash-tag technology, hybridization to mirBase 19 (M19) microarrays, data processing, and statistical analysis, all for a low flat fee. Please see ORB’s microRNA microarray page for further details about the mirBase 19 microarray and pricing information.

ORB's process begins with removal of cells by high-speed centrifugation followed by filtration through a 0.45-micron filter to ensure that the biofluid is cell-free. To monitor the quality of the isolation, labeling, and hybridization procedures, a set of synthetic microRNAs is spiked into each sample. An ABI Taqman real-time PCR assay is used to measure the levels of the spiked microRNAs as well as 2-3 endogenous ubiquitous microRNAs. The samples that pass this test are then labeled and hybridized to M19 microarrays. Signal from the synthetic spike, low background hybridization, specificity of hybridization, and reproducibility are all monitored for each microarray using various control probes.

ORB’s standard data analysis package includes quality control report, log-transformation, normalization, and spot averaging. Data presented in an Excel workbook includes detection/ non-detection calls, ANOVA, hierarchical clustering, and principal component analysis. MicroRNA pathway analysis and/or correlation of microRNA data to gene expression data is available to interested customers. Additionally, ORB provides a complete biomarker discovery package including predictive modeling services customized to clients’ research interests and goals.

Related services

GLP compliant validation and optimization studies using real-time qPCR assays are available to confirm initial circulating microRNA targets. ORB also offers a broader and more comprehensive interrogation of small RNA expression in biofluids using small RNA sequencing. In addition, ORB also provides sequencing-based profiling of exosomal mRNA and Luminex-based multiplex immunoassays compatible with many types of biofluids (please follow links below).


Exosomal Long RNA

Exosomal Small RNA

Immunoassays

 

Sample submission information - MicroRNA microarray

Accepted sample types

  • Plasma
  • Serum
  • Cerebrospinal Fluid
  • Lymphatic Fluid
  • Lavage
  • Ascites Fluid

Sample submission guidance

Table I. Biofluid requirements for microRNA profiling.
Sample Type Fraction Required volume (ml)

Cell-free biofluid (e.g. plasma)

Total 0.325
Cell-free biofluid (e.g. plasma) Microvesicle/
exosome
1.0
Whole Blood Total 1 PAXgene tube
ORB provides RNA isolation services from a wide variety of biofluid samples. ORB is a Biosafety Level 2 (BSL-2) laboratory, and can accept samples which contain agents associated with human disease.


Table II. Sample submission guidance for MicroRNA microarray platform.

Sample Type Sample Preparation Protocol Sample Submission Checklist
Plasma Biofluid - low input
Serum and other cell-free biofluids Contact us for sample-specific recommendations Biofluid - low input
Bronchoalveolar lavage fluid Biofluid - low input

 

Whole blood

 

PAXgene tubes

 
Contact us today for a complementary consultation regarding your biofluid microRNA profiling study.

Example studies using ORB’s proprietary microRNA microarray

Inflammatory Bowel Disease
 
Figure 1. Heat map for significant (FDR<0.05) and differentially regulated (2 fold or greater) microRNA from serum samples from patients having Crohn’s disease, ulcerative colitis, rheumatoid arthritis, or heathy subjects.
 


Crohn’s disease (CD) and ulcerative colitis (UC) comprise the major types of inflammatory bowel disease (IBD). In the pursuit of biomarkers, over 160 unique loci have been identified with genome-wide associate scans (GWAS) that correspond to increased susceptibility to IBD. Dr. Geoff Krissansen’s laboratory at the University of Auckland employed ORB’s microRNA profiling capabilities with human serum samples to identify circulating microRNAs associated with active CD and UC. ORB performed RNA isolations from patients’ sera samples, microarray analysis with multispecies miRBase v. 15 microarrays, and validation of the expression levels of selected microRNAs with ABI Taqman assays in multiple cohorts of patients, see Figure 1.

Examination of microRNAs with ORB’s proprietary microarray v. 15, which is capable of detecting as little as 20 attamoles of synthetic microRNA, and subsequent data analysis revealed that 168 microRNAs were up-regulated by greater than 2-fold in serum of colonic CD, UC, and rheumatoid arthritis (RA) patients with active disease versus healthy control subjects.

A subset of 20 microRNAs were chosen from the original group of microRNAs for Phase II analysis with real-time quantitative PCR (RT- qPCR). Phase II confirmation results identified 5 microRNAs to be significantly up-regulated in at least one disease group. The validation in Phase III was expanded such that cohorts of disease patients included varying levels of disease activity and the number of healthy control patients was increased by 5-fold in order to further validate selected microRNAs with RT-qPCR.
 
Figure 2. Box and Whisker plot of top 4 significant and differentially expressed microRNA in serum samples.
Krissansen and colleagues demonstrated both miR-595 and miR-1246 were significantly increased (P < 0.001) in CD, UC, and RA patients in comparison to healthy controls, see Figure 2, as well as in CD and UC patients with active disease as compared to inactive disease, thereby revealing miR-595 and miR- 1246 as biomarkers useful to patient stratification. (Krissansen GW et al., 2015).

Pancreatic Cancer
Pancreatic cancer persists as one of the most lethal neoplasias, largely due to asymptomatic natural pathology until the disease has advanced to later stages. Prognostic biomarkers, e.g. cancer antigen 19-9 (CA 19-9) and oncogenes like KRAS and p53, show insufficient specificity and sensitivity for the early detection of pancreatic cancer. On this basis, researchers from Valley hospital employed ORB’s microRNA profiling services to query for circulating markers in order to develop a blood-based diagnostic capable of separating patients with Stage IIA or IIB pancreatic ductal adenocarcinoma, high risk controls with potential inherited genetic susceptibility, and healthy volunteers.

This multiphase biomarker discovery initiative constituted evaluation of 1,220 mature microRNAs using ORB’s miRBase version 16 microarray and subsequent validation analysis using ABI Taqman assays for a subgroup of microRNAs.
 
Figure 3. The microarray signature profile of circulating microRNA in pancreatic cancer patients and healthy controls. Heatmap colors represent increase (red) or decrease (green) relative to the median signal.

 

ORB isolated low-molecular weight RNA from plasma samples and then performed microarray experiments which identified 31 candidate microRNA sequences that were differentially expressed in pancreatic cancer patients versus healthy controls, see Figure 3. RT-qPCR assay of an independent set of patient samples showed that miR-642b-3p, miR-885-5p, and miR- 22-3p when evaluated together have the potential to a provide a high level of diagnostic accuracy for early-stage pancreatic cancer (Ganepola G et al., 2014).

 

References

  1. 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.
  2. 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.