Dear friends and fellows,
It will be great to know your experiences working with extracellular/circulating or exosomal miRNAs. We are facing different problems starting from sample isolation to data analysis during our work with extracellular miRNAs. If we share our experiences regarding these issues, it may help other researchers to take right decisions about their work. Thanks in advance.
You can add your problems and suggestions in the following categories-
-- Sample preparation
-- Extraction methods (specific kit and challenges)
-- Measuring concentrations (problems and solutions)
-- Data analysis (normalization method)
Dear colleague(s),
Yes, there may be many obstacles in this kind of research to be resolved.
1) Complex origin of extracellular miRNAs - different sources of origin difficult to ascertain. As alternation in one kind of source cells may affect the whole results or in other "compartments", it is very complicated for the interpretation of results. For example, in plasma/serum/urine, how to distinguish the contributors? We discuss it here http://www.ncbi.nlm.nih.gov/pubmed/25997972.
2) Collection of samples - a) Is it made in the same part of day to overcome effects of the circadian system? What about other external factors.
b) Stabilization of samples - how to stop RNases? Diiferent stabilization tubes or additives. Low RNA concentrations for NGS and (micro)array techniques, compromised CTs (solution?).
3) Isolation techniques - supernatant fractions, exosomal fractions, exosome-depleted fractions, (ultra)centrifugations, different isolation kits leading to different results and enrichment/depletion of some miRNAs. Precipitation and centrifugation of samples - after storage of frozen samples not discarding the protein precipitates with potentialy bound miRNAs. Different diagnostic potential. We discuss it here http://www.ncbi.nlm.nih.gov/pubmed/26639242.
4) Normalization - lack of stable and verified endogenous controls, testing different approaches (e.g., geo-mean normalization).
5) Lack of comparable data and international validation.
6) and possibly many other which would be discussed here.
However, the lack of knowledge is a great space for the research.
Good luck.
Ludek
Dear Dr Luděk Záveský,
Thanks for your contribution. The addressed questions are important and publications you suggested is worthy to read.
One of the important issue is Data Normalization. Well, when you are analysing more than 100 expressed miRNAs global normalization or geo-mean normalization work fine. But when you are working only with few candidate miRNAs this approach may not work. What do you think about adding SPIKED-IN miRNA during denaturing phase.
I am thinking about the obstacle that we may faced in later stage, for example clinical routine test for diseases. As you are working with extracellular miRNA regarding gynaecological cancers, it is expected that you will develop a reliable biomarker for this particular disease which can be used for the detection of the disease with ease.
Is there any unpublished challenges that you faced during your work with extracellular miRNAs? For example, when I was trying to isolate exosome from follicular fluid using Exoquick Kit, the resulting pellet was hard to dissolve with Qiazol Lysis buffer (miRNeasy mini kit). But it become easy when you dissolve the pellet first with 100-200 ul water and then add Qiazol lysis buffer.
Thanks again for sharing your experiences.
have a good day.
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