|From:||yichao wu |
Dear Dr. Farsinejad,
Thank you very much for your comments. You may also have received another email I wrote to histonet just now.
I doubt seriously that our fixative of acetone-methanol (which maintain much antigens excellently) would destroy RNA after incubation for overnight.Another choice for me is Bourin-HgCl2 fixed paraffin-embeded sections.Is it possible?But I'm afraid the low PH (about 4-5)would destroy RNA too.
I wonder how long would you incubate the microdissected tissue with proteinese K? and how is the concentration? I learned from Qiagen protocol that incubation with 20mg/ml PK for 10 minutes at 55 degree.But I learned from several articles that the incubation would be overnight.
Another problem I care about mostly is how would the process of making paraffin-embeded sections destroy RNA? I believe formalin fixation would protect RNA,but How about other steps? From the incubation at 60C after sticking to slides to the deparaffinization.
Any comments are welcome.
Yichao WU, Ph.D candidate
Research Institute of Nephrology,305# East Zhongshan Road,Jinling Hospital,Medical School,Nanjing University,Nanjing, 210002,P.R.China
Another question is,since the frozen sections are not as good as paraffin-embeded ones in morphology.I wonder who have successfully acquired RNA for RT-PCR from laser microdissected tissue on paraffin sections?I have tried and tried and failed.The kits I used are "Qiagen Rneasy Mini kit"(I have no "Micro kit" at hand) and one from arctrus and one from Gentra called puroscript.And after Dnase digestion,it seems that I have not acquired any RNA from paraffin embeded sections.The fixation is acetone-methanol(1:1).I wonder if such fixation would destroy RNA? And I wonder if heating sections after paraffin embeding is destroyable? How are other procedures should be followed for microdissection on paraffin embed sections? Pls help me out.Thank you very much!