I am trying to build an miRNA profile from a number of cancer tissue/normal samples via RNAseq. What I needed to know is, how much is it essential to sort the tumor or normal population of the tissue by a technique such as laser microdissection, prior to NGS?
Laser Capture Microdissection (LCM) is very important tool to compare between two different profiles of gene expression in different tissues. For instance, I have used these techniques to explore the difference in gene expression between normal tissue and nematodes infected tissues and identify different expressed gene in both tissues. I the same manner, you can use it to different expressed genes through exploring miRNA profiles from a number of cancer tissue/normal samples. It is really very useful technique. Good luck, I hope this answer could help
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HI for a similar reason I had to do the same, I needed some extremely pure cell populations so I could analyse them. the best option you have is PALM from Zeiss if you have access to one your problems are over. with it you can cut areas of the cells or whole cells out of tissue and zap them into an eppendorf without touching anything so you are 100% sure that there is no contamination whatsoever. it took me about 3 months to master but it works a treat. Good luck
Hi all,
thank you very much for your answers. However, what i needed to understand is that if i have tumor and adjacent normal tissue available for rna seq based analysis. Would it be still advisable to go for microdissection of specific portions for further analysis? Is there a cut-off on the average tumor content essential for a better ngs based analysis?
Briefly, if you already have both tissues precisely isolated, then you only can just go ahead and isolate the total mRNA from both tissues for further analysis. GOOD LUCK
In tumors you almost always a mixture of cancer and normal cells. Laser microdissection adds yet another level of complexity.
Still - you partly answered yourself already. Make subsequent portions, sequence them using a fraction of Illumina lanes (amplification can be done now even from a tiny amount of material) - and after mapping and counting check the level of your preferred cancer marker genes. Then you will have the idea what portions are (almost) proper tumor or healthy tissue. It will cost a bit more perhaps, but it will be a very decent methodology - you can try to write a separate methods paper (Biotechniques. etc) if you succeed with that. I would be curious.
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