total RNA extraction from infected protoplasts - (May/12/2006 )

Hi Guys,
I’m working in virology lab and from time to time I have to struggle with total RNA isolation from protoplasts infected with virus.
Unfortunately, my RNA is very often slightly degradated, even if I am using typical procedure to avoid this effect i.e. DEPC treated H20 is used for preparation of all buffers, I’m wearing gloves, am not sneezing and crying close to my samples, however there is sometimes great temptation to do that 
So, I am asking you for some advices how to avoid degradation of RNA?
I have to specify the procedure which I am using:
1) Protoplast RNA isolation buffer:
50 mM Tris-HCl (pH 7.5)
100 mM NaCl
1 mM EDTA
1% SDS
0.3 mg/ml bentonite
2) Twice phenol/chloroform extraction pH 4.2
3) Precipitation with LiCl 8M pH5.2; 2h -70 C
4) Precipitation with NaoAC pH5.3 in ethanol; over night -70C
I know that the clue of the degradation is somewhere in extraction procedure, because separation on denaturizing gel is O.K. (checked by positive control, viral RNA).
Any ideas?

hi
i think that most of the degradationoccurs during the lysis step. Unfortunately, and appart if you can isolate chloroplast in a quick way, this step seems to maintain.
You can keep your RNA in formamide if they are for northern blots. If they are for RTPCR, DEPC water is better.
Btw, do you use RNAlater or sthg equivalent during isolation steps?

No, I use just these solution which I've mentioned.
Nothing more...and I do not store RNA for a long time, because I usually do Northern blot in the same week when I do RNA extraction.
So, you suggest that most of the RNA degradation occurs during lysis, but I use bentonite which is suppose to absorb all RNases and prevent degradation...hmm

hi
so if that's for northerns, i would recommend you to keep them in formamide, who is better than H2O for avoiding RNase activity. Ok there are RNase free water solded, and DEPC treated water, but in fact, contamination appart from water may occur and contribute to RNA degradation.


The key step in the RNA extraction, seems to be the lysis step. In that step all is quite unorganized, and if the lysis is too slow, RNAses are allwed to do their job (which is quite fast).

I reformulate my qestion :
do you isolate protoplasts and then start your RNA extraction with mentionned buffer? or do you do the isolation with the buffer too?

I isolate mesophyll protoplasts in one day and infect these protoplasts with RNA virus. Then, I place the freshly transformed protoplasts in protoplast culture medium for 24 h, and after that I isolate total RNA (from protoplasts + viral RNA). This is the system for efficient viral infection allowing me to check which cell components might affect viral life cycle.

Question: what is the concetration of formamide that you use to store samples?

hi
regarding your question : it's stock solution, so 100%formamide.

For your exp : i would change for trizol or tri reagent which is cheaper. you'll get better results as i think.


RQ : fixed post, thanks ful to mfdenko who raised my mistake...

formamide should be used "neat" (100%).

formaldehyde is 37%.

Thanks Guys...I will try to convince my boss to buy TRIzol ;-)
I' m also convinced that it would help a lot