Quantification of microRNA cancer biomarkers in serum/plasma using Droplet Digital PCR (ddPCR) miRCURY LNA™ Universal RT microRNA PCR
Dr. Manuela Ferracin
Dr. Manuela Ferracin
is a postdoc in Prof. Massimo Negrini's lab at the Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.
What is the main focus of your research?
Our goal is to quantify the amount of specific cell-free microRNAs in the blood of cancer patients (using both serum and plasma) and to compare patients’ microRNA levels with that of healthy people, in order to identify candidate cancer biomarkers. We have been studying microRNAs since 2004. Over the past ten years, we have performed microRNA expression profiling of many different human cancers. Recently, we decided to decipher the cancer circulating microRNA profile because cell-free microRNA biomarkers would be easily assessable and have the potential to become a minimally invasive diagnostic tool.
What is the aim of your project involving microRNA ddPCR?
Circulating microRNAs are present in blood at extremely low concentrations. Thus we used currently available high-throughput technologies (microarray, NGS) to identify microRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. The aim of this project was to assess the absolute expression level of specific microRNAs using droplet digital PCR (ddPCR) technology (QX200 system, BioRad).
What technologies did you use?
We compared the miRCURY LNA™ Universal RT microRNA PCR System from Exiqon with another commercially available microRNA PCR system, using both systems with ddPCR technology for specific microRNA detection in human biofluids. Although based on different detection chemistries, both systems provided comparable results on the QX200 ddPCR system (Miotto et al., 2014
). Specifically, we used EvaGreen dye-based ddPCR with Exiqon miRCURY LNA™ Universal cDNA synthesis kit II and microRNA LNA™ PCR primer sets.
What were some specific challenges in your experiments?
EvaGreen chemistry had never been tested on a ddPCR instrument for microRNA quantification. We decided to use this chemistry together with Exiqon LNA™ PCR primer sets to increase the assay specificity. We tested several primer pairs on serum and plasma samples and all of them worked fine in serum and plasma samples with EvaGreen droplet digital PCR supermix.
Why did you choose Exiqon’s miRCURY LNA™ Universal RT microRNA PCR system?
The amount of RNA that can be extracted from plasma and serum samples is low. In this context, it is of great importance to be able to quantify any desired microRNA using individual assays and ddPCR technology, without having to do a microRNA-specific reverse transcription. Therefore, a universal cDNA system, like that developed by Exiqon, paired with a specific LNA™-based assay made the Exiqon system very attractive.
What are the future perspectives for this research?
We plan to go on using the Exiqon miRCURY LNA™ Universal RT microRNA PCR System and droplet digital PCR technology for our circulating microRNA quantification.
Where can we read more about your studies?
We published a methodological paper at the end of 2014 ( Miotto et al., 2014
). We applied this methodology in our microRNA biomarker research that finally led to a new publication ( Ferracin et al., 2015
Protocol Protocol for microRNA PCR profiling using the Exiqon miRCURY LNA™ microRNA PCR System with the QX200™ Droplet Digital™ PCR System.