Hi All,
I need your suggestions on the laser capture.
I have been trying to isolate RNA from colonic epithelial cells of Postnatal day 1 mouse by laser microdissection without any success. Here is what I did:
1. Prepared 18 microns fresh frozen sections with PEN membrane slides and stored in -80 freezer less than four weeks before laser capture.
2. on the day of laser capture, leave slides 30s in 75% ethanol, 30s in DEPC-treated water, 30s in histogene staining solution, 30s in DEPC-treated water, 30s in 75% ethanol, 30s in 95% ethanol, 30s in 100% ethanol, and 5 minutes in xylene.
3. after slides are dried for 2-3 minutes, use capsure HS LCM caps to perform laser capture.
4. use picopure RNA isolation kit to isolate RNA. I usually combine extraction buffer from two caps for the RNA isolation.
I either got very low RNA (around 100ng), or no RNA. I could not figure out whether it is due to RNA extraction or RNA degradation. It usually takes me around 1 hour to perform the Laser capture on a cap in order to get as many tissues on a cap as possible. Is it too long? Also according to the protocol, I only added 10 ul extraction buffer to the capsure-extracsure assembly for lysis. I saw the extraction buffer stayed on the wall of the well instead of flowing down to the bottom of the well where the tissues are located after I piptted it into the well . Is this a problem?
I greatly appreciate your suggestions!
Wenyan
Hello:
When I started to isolate RNA from laser microdissected samples I always put 10% RNA later in all the solution that I used to treat the sample and I always put a piece of dry ice near the sample while I was doing the microdissection to keep it cool during the all the process.
hope this helps, good luck
Vir
HI Virginia,
Thank you for your suggestions. Do you mean you add RNA later in the water and 70% ethanol that used for processing the cryostat sections?
Wenyan
Hello:
Yes, I added RNA later in all the solutions that were in contact with the sample..
Best
virginia
Recently, a new book chapter has been published, which contains a lot of useful tips:
Chapter Laser Microdissection Workflow for Isolating Nucleic Acids f...
Thank you for this Q&A! I have been struggling tremendously with this. Also does anyone have a better stain to use with FFPE tissue sections other than the Paradise staining set? I can't see anything with this stain. It is all blue and grey....
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