microRNAs as disease biomarkers microRNA PCR Services
Dr. Wojciech Fendler
Dr. Fendler Wojciech
works in the Department of Pediatrics, Oncology, Hematology and Diabetology at the Medical University of Lodz, Poland. He is interested in the application of microRNAs as biomarkers in disease and has performed microRNA profiling using Exiqon Services.
What is the main focus of your research/the research conducted in your lab?
We coordinate a large, nationwide database of patients with diabetes caused by single gene mutations or complex genetic syndromes that manifest with diabetes. Other research interests include childhood malignancy and its genetic determinants as well as tuberous sclerosis.
How did you come to be interested in microRNAs?
A collaborative study with Dr. Dipanjan Chowdhury from the Dana Farber Cancer Institute showed the potential for microRNAs as biomarkers of disease, which I plan to use in the field of diabetology/genetics.
Which experiments had been performed leading up to this project?
We have analyzed animal serum profiling samples from a radiation viability experiment with different levels of exposure. Profiling was performed using Exiqon qPCR panels and evidenced robust and consistent microRNA patterns with potential clinical use.
What is the aim of your current project?
To determine single-gene-dependent regulatory patterns of microRNA expression in the context of diabetes.
We specifically wanted to find out if a single-gene knockout would provoke specific microRNA patterns in serum by analyzing samples from patients with different genetic backgrounds.
What was your previous experience with microRNA analysis/qPCR?
We had not performed any qPCR profiling ourselves, but I have been involved in the analysis of microRNA profiling data from a fellow collaborator.
How did you perform the experiments and analyze the results?
Our study included serum from four separate groups of patients and healthy controls. The RNA isolation and microRNA profiling were done by Exiqon Services. We used the normalization protocol from Exiqon and followed through with standard data mining procedures using Multi-Experiment viewer and Statistica software.
What were some specific challenges in your experiments?
The most difficult part of the analysis was the normalization of profiling data from different experimental batches. Serum samples were submitted in two lots of 20 and 40, which required a unified normalization standard later on. As there are no suitable reference or housekeeping genes in serum, this was performed using the Exiqon protocol for normalization against universally expressed microRNAs with stable concentration across the samples from both batches.
How did you overcome them?
We used Exiqon’s quality control procedure and received full support in matching differently normalized samples.
How do you feel about your results so far?
Very satisfied both with the quality of the data and customer service.
Why did you choose Exiqon’s miRCURY LNA™ Universal RT microRNA PCR system?
It was based on recommendations from my colleagues and the good working experience of my collaborators. The high quality of the data and very good reproducibility of results convinced me to use the product.
What do you find to be the main benefits of the Exiqon microRNA qPCR service?
The best thing about the service was the very good customer support throughout the whole process of experiment planning, execution and analysis.
Why would you recommend Exiqon and/or Exiqon’s microRNA qPCR system to colleagues?
Excellent technical and scientific feedback allows researchers from different fields to incorporate miRNA experiments into their study protocols.
What would be your advice to colleagues about getting started with microRNA qPCR analysis?
Plan everything ahead and consult Exiqon advisors for all potentially problematic issues.
What would you recommend to others regarding sample preparation/normalization/data analysis?
Read the instructions thoroughly and use the recommended methods.
In your opinion what is the most important factor for a successful microRNA experiment?
Thorough planning and a good understanding of statistical methods used to analyze the resulting profiling data.
What are the next steps in the current project and how do you plan to perform them?
To evaluate the differentially expressed microRNAs from the serum profiling and elucidate their tissue of origin, we have performed in vitro
gene silencing of two transcription factors. We used the HepG2 and HEK 293 cell lines with the siRNA knockdowns performed as a courtesy in the Nencki Institute of Experimental Biology in Warsaw.
What are the future perspectives for this research?
After the cell-line validation experiment , we may look into regulatory effects of specific microRNAs with potentially therapeutic applications.
When and where will be hear /read more about your studies?
Most likely during late 2014 if everything proceeds as planned with the in vitro