MicroRNA (miRNA) are short, non-coding, single-stranded RNA molecules that regulate gene expression at the post transcriptional level. They are known to play a critical role in multiple biological processes including proliferation, differentiation, apoptosis and hematopoiesis. Dysregulation of miRNA expression therefore is associated with a number of pathological conditions such as inflammation, cardiovascular diseases, neurological disorders and several types of cancers. miRNAs are also found to be extremely stable in body fluids such as serum, plasma, urine, breast milk and saliva. Extracellular miRNAs are either packed into exosomes or loaded into high-density lipoproteins, or bound by AGO2 proteins outside on vesicles. These modes of action protect miRNAs from degradation and improve their stability. Because of their stability in body fluids, miRNAs are gaining importance as biomarkers in liquid biopsies. Real time qPCR technologies reliably and accurately detect and measure miRNAs levels from body fluids. However, since no single miRNA is known to be constitutively expressed in body fluids, normalizing real time qPCR data for comparing miRNA expression levels in different samples continues to be a challenge.
The study described in this webinar demonstrates a systematic methodology to identify and verify stably expressed miRNAs in body fluids. The stably expressed miRNAs are then used to normalize miRNA expression profiles in plasma. A workflow is illustrated for identifying miRNA biomarkers in plasma using the TaqMan Human Advanced miRNA Panels on 96-well plates, Taqman Array Cards (TAC), and Open Array plates. These different formats provide throughput flexibility for miRNA biomarker discovery research programs.
- How to identify stably expressed/endogenous miRNAs in serum and plasma
- How to use spike-in controls to normalize miRNA expression in difficult samples
- Workflow to identify normalizers in serum and plasma using TaqMan Array Cards, Plates and OpenArray
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R&D Staff Scientist, Sample Preparation, BioSciences
Thermo Fisher Scientific
Emily Zeringer has worked in the R&D department at Thermo Fisher for over 15 years. During her tenure, she has led and contributed to many projects including sample preparation and analysis of nucleic acids (including real-time PCR) from a wide range of sample types, library preparation for sequencing, and purification and analysis of exosomes. In recent years, her efforts have shifted to the development of high-throughput and automated DNA/RNA extraction methods from clinical samples (such as biofluids) for use within larger analysis workflows including pharmacogenomics, pathogen/microbial detection, disease studies and biomarker studies.
Harita Veereshlingam, PhD
Senior Product Application Scientist
Thermo Fisher Scientific
Harita Veereshlingam is a senior product application scientist at Thermo Fisher. She has over 10 years of experience in product development with an emphasis on real-time qPCR assays for gene expression and miRNA analysis. In her current role, Harita is responsible for product portfolio growth through the development of novel applications, customer collaborations, technical notes and webinars. She lives by, and is a strong proponent of, sustainable living. She has a PhD in Molecular Biology from the University of North Texas.