Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)
Moderators: mchlbrmn, Abhijeet Bakre
Could anyone tell me how to get good quality RNA from tumour tissues embedded in OCT.Both Tri reagent and Quaigen RNeasy give degraded RNA.Could some one suggest any other alternative prtocol to get rid of OCT. [quote][/quote]
Iam working in the area of cancer genetics.Iam interested incomparitive genomics and gene expression studies.
- Posts: 1
- Joined: May 15 2004 7:06 am
- Location: Saudiarabia
I extracted RNA with RNeasy and it worked well. Do not hesitate to ask Qiagen Technical Service - they always have a good advice.
Turbett & Sellner (1997) use Phenol-Chloroform and have problems with subsequent RT-PCR (PMID: 9458390) while McDonald et al. use Trizol and habe no problems with RT-PCT (I didn't find the paper in PubMed).
Is it possible that your RNA is already degraded and/or the tissue was not frozen properly ?
- Posts: 13
- Joined: Apr 15 2004 6:59 am
- Location: Germany
As Ivo... I'd look for RNA degredation in your tissue or perhaps from your OCT slide. (And call Qiagen - they've got great tech support)
I do Laser Capture Microdissection on OCT frozen human tissues from the operating room. The RNA is often degraded, before I can process it in the lab.
I have enough tissue to both freeze in OCT and to flash freeze in liquid Nitrogen.
I assume you may be staining cryostat cut OCT sections prior to RNA prep?
Or perhaps your slide is getting wet from the atmosphere (a problem for me in the humid Midwest USA, perhaps not in Saudi Arabia?!) and RNases are having a chance to degrade your RNA.
My cryostat cut OCT sections, without staining, scraped into an eppendorf tube and RNA prep'd, seem to be about the same quality (good or bad) as the liquid nitrogen frozen tissue.
Several LCM users warned me to take great care in my slide staining, to minimize the time and possible RNase activation/contamination. And fortunately my stained slide RNA quality is about the same as unstained slide OCT sections.
My main point is, all of my bad OCT slides led back to a bad liquid nitrogen frozen tissue. Tissues apparently degrade as soon as they are clamped off in the operation. I'd minimize this time (I'm down to about 10 minutes from receiving the OR call to having everything frozen), and also think about your OCT slide processing.
Good Luck to us all,
- Posts: 42
- Joined: Nov 04 2003 12:16 pm
- Location: Washington Univ St. Louis
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