Strategies to normalize miRNA expression in serum and plasma.

Presented By:
Emily Zeringer - R&D Staff Scientist- Sample Preparation BioSciences - Thermo Fisher Scientific

Speaker Biography:
Emily has worked as part of R&D at the Austin site for over 15 years. During her tenure, she has lead and contributed to multiple 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. Recently and for the past several 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 ranging from such as Pharmacogenomics, Pathogen/Microbial Detection, Disease studies and Biomarker studies.

Co-Presented By:
Harita Veereshlingam, PhD - Senior Product Application Scientist

Speaker Biography:
Harita Veereshlingam is a senior product application scientist in the genetic science division at Thermo Fisher Scientific. 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.

Webinar:
Strategies to normalize miRNA expression in serum and plasma.

Webinar Abstract:
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. In this study, we demonstrate 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. We illustrate this workflow 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.

Learning Objectives:
-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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[shwetashweta]

How can we statistically calculate endogenous control? Could i use Kruskal Wallis test.
I have 5 reference gene and i found one miRNA that was not significant but remaining 4 was significant. i did with Kruskal Wallis test.

[angelemmanuel]

How to normalize my data results with my endogenous control?