Introduction
Serum, plasma and other bodily fluids contain microRNA that can serve as biomarkers in many disease states including cancer ( Chen, X et al., 2008 , Mitchell, P.S. et al., 2008 ). 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 serum using our custom Sanger15 mammalian microRNA array. 174 human microRNAs were detected in plasma from normal volunteers in our preliminary experiments. For a comparative analysis of the number of microRNAs detected from plasma vs. human universal tissue pool see Figure 1 below.
Figure 1: MicroRNA content of plasma (normal volunteers) vs. Human Tissue Pool
ORB scientists have validated five miRNAs detected by microarray in serum samples using qRT-PCR experiment with TaqMan miRNA assays developed by Applied Biosystems which are considered as the Gold Standard in the validation of microarray data. Correlation of microarray expression results with qRT-PCR results is shown in Figure 2A-E.
Figure 2: Correlation plots of microarray probe intensites and real time quantitative PCR Ct values for five different microRNAs detected in serum from normal volunteers.
Procedures
ORB requires at least 1 ml of serum or other biofluid per sample to obtain the required amount of RNA for microRNA profiling. ORB's process begins with removal of cells by high-speed centrifugation followed by filtration through 0.45-micron filter to ensure that the biofluid is cell free. To ensure the quality of our isolation and labeling procedures, we spike a set of synthetic microRNAs in to each sample. We also perform a quality control test using ABI Taqman real time PCR on the samples to detect the presence of the synthetic spiked RNAs and two endogenous ubiquitous microRNA. The samples that pass the QC test are then labeled using the sensitive Genisphere - Flash tag labeling system and hybridized to mirBASE version 16 multispecies microarrays.
Data Analysis
Our standard data analysis package includes quality control report, log-transformation, normalization, and spot averaging. Data presented in Excel workbook includes detection/ non-detection calls, ANOVA, hierarchical clustering, and principal component analysis (where applicable). MicroRNA pathway analysis and/or correlation of microRNA data to gene expression data is available to interested customers.
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