|From:||"Gagermeier, James" |
First time on the Histonet -
These are questions that pertain to laser microdissection on lung tissue using a Leica AS LMD with the goal of performing microarray. If anyone can answer one, all of some of these questions I appreciate your input.
1) In staining tissues with nuclease free alcohols - Arcturus recommends that these alcohols be changed every 4 slides. I have noticed that using the alcohols for 10-12 slides does seem to lead to more difficult capture of the cells as they seem to adhere to the slides more tightly and do not drop into the caps. In an attempt to be economical, does anyone have recommendations for how many slides can be stained before changing alcohols/water is necessary?
2) I have ordered glass foiled slides to capture the cells, as opposed to glass slides, as I have been told that the foiled slides retain H2O less avidly - furthermore, it would seem that cutting through foil would naturally be easier than through the slides.
3) I have also read that if glass slides are used, that these too should be cleaned in DEPC-treated solution, autoclaved, and even RNAZapped. This seems a bit much - is this true?
4) In regards to the Leica system - is amplification of tissue almost universally necessary to obtain adequate amounts of RNA (lower limit .2 ug) to perform a Codelink platform microarray? Initially, I obtained a yield of about .3 ug from microdissected tissues. This yield was obtained from capturing a total area of 495,000 um2 of tissue from about 10 individual captured pieces of tissue (ranging in size from 9,000 - 180,000 um2). However, in subsequent sessions of microdissection, similar areas of tissue gave me a total of 40-120 nannograms of RNA - which will obviously need amplified. Should I expect to amplify most of the time?
5) In lung tissue, what is the minimum tissue area that needs to be captured ( if anyone wants to give me their thoughts) that is sufficient, then, to avoid amplification?
6) No number 6.