total RNA from BSE infected WBC for microarray and qPCR - (Sep/30/2005 )

Dear All

I have been trying to isolate total RNA from white blood cells for a while with little success. The case is complicated by the fact that the original blood was from BSE infected animals and so the method I use must involve the addition of at least 4M guanidinum isothiocyanate to ensure that the samples are safe for me to handle. I have been using a modified Chomzynski method which involves the used of RNAbee as the initial Trizol 'like' reagent and then a series of phenol:chloroform and isoamyl alcohol washes before washing the minute or sometimes non-existent pellet in 80% ETOH and then finally re-suspending in nuclease free H2O. Nanodrop readings have tended to show good ratio (1.7) and between 10 and 50 ng/µl RNA however when I run the samples on an Agilent Bioanalyser pico chip all I can see is what is potentially a 18s related bump but that is it.

As another part of this project I have used this isolation method with great sucess on the speens of these same animals so I know that the method does work.

The mice that the blood came from are very small so the total amount of WBC recovered was also very small. Once the blood was drawn the WBC were isolated immediately by density gradient centrifugation and the pellets have been maintained at -80C until I added in the RNAbee. As the pellets are so small they disperse into the RNAbee immediately with a little help from a gentle pipetting. Under cat3 conditions all the other steps are carried out where possible on ice and I work as fast as possible.

I am now almost at the stage of giving up on these samples as I need good quality RNA to take forward to microarray analysis, but before I do has anyone got any suggestions?

Thanks in advance

Auriol

Dear Auriol,
Before going on the microarray experiment you check the 260/280 ratio (1.8-2.0) and also 260/230 ratio which will be 2.0 or beetween 2.0-2.3. some times when phenole remains in the RNA samples its gives false reading which show good 260/280 ratio.












Dear All

I have been trying to isolate total RNA from white blood cells for a while with little success. The case is complicated by the fact that the original blood was from BSE infected animals and so the method I use must involve the addition of at least 4M guanidinum isothiocyanate to ensure that the samples are safe for me to handle. I have been using a modified Chomzynski method which involves the used of RNAbee as the initial Trizol 'like' reagent and then a series of phenol:chloroform and isoamyl alcohol washes before washing the minute or sometimes non-existent pellet in 80% ETOH and then finally re-suspending in nuclease free H2O. Nanodrop readings have tended to show good ratio (1.7) and between 10 and 50 ng/µl RNA however when I run the samples on an Agilent Bioanalyser pico chip all I can see is what is potentially a 18s related bump but that is it.

As another part of this project I have used this isolation method with great sucess on the speens of these same animals so I know that the method does work.

The mice that the blood came from are very small so the total amount of WBC recovered was also very small. Once the blood was drawn the WBC were isolated immediately by density gradient centrifugation and the pellets have been maintained at -80C until I added in the RNAbee. As the pellets are so small they disperse into the RNAbee immediately with a little help from a gentle pipetting. Under cat3 conditions all the other steps are carried out where possible on ice and I work as fast as possible.

I am now almost at the stage of giving up on these samples as I need good quality RNA to take forward to microarray analysis, but before I do has anyone got any suggestions?

Thanks in advance

Auriol
[/quote]

Dear Awadh

Thanks for your reply however in the years that I have been working with these BSE infected samples RNA isolation using this method rarely results in a perfect 2 ratio and I have found that ratios of 1.6 to 1.8 are more common with this particular sample type. I have found that when I carry out a cleanup step with RNeasy columns the ratio improves marginally, this step removes some of the phenol contamination and so I routinely carry out this cleanup step.

All my previous experience with these samples has been using the spleens and not the WBC and my primary problem is the lack of anything other than 18s RNA in the whole blood cell samples as I cannot take these samples forward to reliably analyse the expressed genes. My general feeling is that the samples are degraded but if anyone has any suggestions re a better method of isolation I would be most grateful.

Thanks

Auriolw



[quote name='awadh' date='Oct 1 2005, 01:07 PM' post='26393']
Dear Auriol,
Before going on the microarray experiment you check the 260/280 ratio (1.8-2.0) and also 260/230 ratio which will be 2.0 or beetween 2.0-2.3. some times when phenole remains in the RNA samples its gives false reading which show good 260/280 ratio.