microRNAs as biomarkers for fertility disorders miRCURY LNA™ Universal RT microRNA PCR
Dr. Sara Larriba
leads the Molecular Genetics in Male Fertility research line of the Human Molecular Genetics Group at the Bellvitge Biomedical Research Institute (IDIBELL) in Barcelona (Spain). She has been using Exiqon's miRCURY LNA™ Universal RT microRNA PCR system for profiling microRNA in spermatozoa from semen samples.
1. What is the main focus of your research?
Our group is interested in a better understanding of the molecular mechanisms that lead to male infertility. Furthermore, we are interested in studying the role of non-coding RNAs in the regulation of spermatozoa gene expression and the potential of microRNAs as biomarkers for fertility disorders.
2. How did you come to be interested in microRNAs?
In recent years, we have been studying differences in sperm transcript profiles associated with the fertilizing ability of spermatozoa, however the potential differences in the small RNA fraction and the role of microRNAs in the regulation of spermatozoa gene expression has yet to be determined.
3. What is the aim of your current project?
Our current project involves the analysis of microRNAs in spermatozoa and their potential as biomarkers for diagnosis and prognosis of individuals with fertility disorders.
4. What were some specific challenges in your experiments?
One of the major drawbacks of the analysis of sperm microRNAs is the low level of coding and non-coding RNAs contained in spermatozoa. Our main challenge was, thus, to reliably detect microRNAs in spermatozoa. We needed to optimize the RNA extraction procedure and additionally use a very sensitive and reliable microRNA qPCR system.
5. How did you overcome these challenges?
In order to optimize the RNA extraction procedure, we first worked with several column based RNA purification kits from different companies and compare the efficiency of total RNA extraction and the quality of RNA. We found that miRCURY™ RNA Isolation kit was a robust and the most efficient method for sperm RNA extraction. Furthermore, in order to obtain as much information as possible from the limited RNA sample amount available we wanted to use a sensitive and reproducible method. We found that Exiqon microRNA qPCR panels were appropriate for our current project.
6. How do you feel about your results so far?
We are very confident about our data using Exiqon panels.
7. What do you find to be the main advantages of the miRCURY™ RNA Isolation kit and miRCURY LNA™ microRNA PCR system?
Exiqon products are based on efficient and easy to use methods. Most importantly when working with samples with low microRNA content, they provide sensitive and highly reproducible results.
8. What would you recommend to others regarding sample preparation, normalization and data analysis?
I think that the first point to consider is to be aware of the availability of the starting material and the level of microRNA expression in your target tissue under physiological conditions. It is important to test several techniques for RNA extraction and microRNA quantification platforms to try to get the most out of your samples. Optimization of RNA input in RT and PCR reactions should be additionally considered. Furthermore, the use of controls, such as the RNA spike-in for RNA extraction and RT reaction and interplate calibrators for qPCR experiments, gives you confidence in your results.
9. When will we hear more about your studies?
We are preparing a manuscript with the results of the current project.