Hello Everyone!
I am trying to extract intact and high integrity RNA from fixed cells. The idea is to sort cells using FACS and from sorted population extract RNA. The samples is mouse tissue cells which is converted to single cell suspension using enzymes. Steps till sorting takes a lot of time and I am hoping to break the protocol down and do RNA extraction second day from sorted cells fixed with formalin or ethanol (or any other suggestions?)
I tried extraction of rna from fixed cells but found it is degraded. I was wondering if I can add proteinase k after formaldehye fixation to remove the RNAse. I found that proteinase k needs 65 degree C for its activation and my RNA sample might get degraded if I heat it to that temperature.
I would like to know :
1- Can ethanol fixed cells be used to extract RNA (I got positive results but not sure if the RNA would be modified by ethanol fixation) ? If yes, then do we need to inavtivate RNAse in ethanol fixed cells?
2- Can formaldehye be used to fix cells and later extract RNA with good integrity?. I would love to see any published protocol or paper.
3- After fixation, I can I protect my RNA from being degraded by RNAse in sample. Would adding proteinase k help? If yes what are the conditions (temp, concentration and pH) for proteinase K to be used to degrade RNAse?
4- Any other suggestions to extract RNA for gene expression studies.
The RNA thus obtained would be used for gene expression studies using qPCR or RNA seq.
Please provide some good suggestions.
Hi Ankur,
Do not expect that proteinase K will help to prevent RNA degradation: it is relatively slow and many RNases are resistant to its action. Opt for approaches based on rapid denaturation of the sample such as Trizol or analogous described in the answers above. These are most useful suggestions.
Try direct sorting into Trizol or RLT buffer, ethanol, or PBS with an RNase inhibitior, or media with RNase inhibitor. If you sort into Trizol LS or RLT you need to extract your RNA on the same day. Do not freeze and extract days later.
These might be also for help in selecting a protocol:
http://onlinelibrary.wiley.com/doi/10.1002/cyto.990110803/pdf
http://www.sciencedirect.com/science/article/pii/S0165022X99000202?via%3Dihub
I would recommend to fix your cells with preserving agents such as RNAlater or LifeGuard Soil preservation solution. You might need to remove most of it before you continue with your extraction as it could interfere with your extraction buffer.
I never used proteinase K but rather beta-mercaptoethanol to prevent my RNA from degradation during extraction. Depending on the protocol I only added 1% v/v and it worked perfectly. You need to work under a fume hood because this chemical smells...
Hi Ankur,
Do not expect that proteinase K will help to prevent RNA degradation: it is relatively slow and many RNases are resistant to its action. Opt for approaches based on rapid denaturation of the sample such as Trizol or analogous described in the answers above. These are most useful suggestions.
the best and more easy way is to microdissect the cells of the interest from 5 um section fixed in glass slide and follow regular protocol for RNA extraction from paraffin using either Qigen or Biored extraction Kit
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression