RNA extraction from laser capture microdissections - how to see low expressed genes from trought RT/qPCR on laser capture m (Mar/13/2008 )
Hi to everybody,
we have a P.A.L.M. laser capture microscopy, we usually extract DNA from paraffined section and we don't have great problems.
The problems occur when we try to extract RNA from frozen or paraffine material.
We have followed the P.A.L.M. protocol, but actually no RNA is in our tubes.
My purpose to obtain enough RNA to see HOX genes (low expressed genes) in real-time pcr reaction.
Is here someone who extracts RNA from laser capture microdissections????
Could you suggest me a protocol to use????
Are some tricks to apply???
thank you a lot it very important for my job.
personally, I'm not working with laser captured microdissections but maybe the amount of extracted RNA is just too low and a preamplification step is the trick.
There is a preAmp mastermix from Appllied Biosystems (part # 4391128) availabe, which they claim doesn't introduce a bias:
"Amplify cDNA targets equally without introducing bias
Analyze mRNA from any precious sample such as laser capture microdissections, needle biopsies, and formalin-fixed paraffin-embedded tissues (FFPE)
Stretch as little as 1 ng of cDNA into 200 real-time PCR reactions for gene expression analysis using TaqMan® Gene Expression Assays
Analyze up to 100 gene expression targets with minimal hands-on time"
but the price is
however, if you are considering this method you should first contact your local ABI sales representative to discuss if it is really the right application and also to get some discount.
You should be careful that the RNA is not degraded, and that you have a sufficient amount. I'm not familiar with the PALM system, but I use a Leica AS-LMD setup to study frozen bone/cartilage, in which the cut out specimen drops into RLT w/B-ME extraction buffer in a eppendorf cap. (Have a look at the attached paper for specific details). Once in the buffer, degradation is not too much of a problem, and can be extracted using RNeasy columns. Where the degradation may become an issue is related to the amount of time that you have your specimen out at room temp. WE noticed that RNA integrity was good until about 15 minutes after removing from dry ice, but degreded precipitously thereafter. You also may have to cut a lot of sections to get a good yield (~30-200 ng), but we usually generate a sufficuent amount of RNA to for Array studies (with amplification as Ned Land suggested) and corroborative Real-Time from the same sample.
As far as for Paraffin sections, you have to make sure you get the paraffin out of the section before you cut it on the LMD. I think Qiagen makes an RNeasy type kit for that too which includes the proteinase K