I'm aiming to extract RNA from laser microdissected samples. Can anyone offer advice or point me to a protocol that works? I want to be able to dissect stained (IHC) cells from a section (I've read frozen works best) and extract RNA from those. Ideally I want the quality and quantity to run on microarrays.
Hope someone can help.
Hello Adam. The choice of method depends upon the material being microdissected and the amount. For RNA frozen works best, but the samples are also susceptible to RNA degradation especially if exposed to aqueous media for too long (this also depends upon the type of tissue) especially during staining.
There are several well tested kits in the market; I have had good experience with Qiagen's RNEasy Micro kits and also with Picopure kits from Life Technologies. It is also possible to use Trizol, however in this case processing a large number of samples can be challenging.
For running microarrays you will very likely have to microdissect a large amount of material. With most tissue 5-10 square mm of collection should give you enough RNA for doing a microarray. Try to find out the minimum quantity of RNA that is required by your microarray facility and then determine how much tissue you have to collect. In my experience 1 square mm of microdissected material from a 10µm thick section can yield between 20-80 ng RNA (again, this is highly dependent on the tissue type).
Hope this helps.
I attached a publication which uses Laser microdissection to isolate tissue for subsequent RNA isolation and microarray analysis. I hope this helps!
Article Identification of novel immune and barrier genes in atopic d...
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