RNA from Sheep and Bovine blood
Posted 08 March 2009 - 03:22 PM
Ive just started working in a group that is researching Johnes disease in bovine and ovine models. Presently blood is being taken once a fortnight and it is being separated into buffy and PBMC and then RNA isolated. The process takes 2 days and so far the RNA is OK but there is always gDNA contamination even after DNase treatment. My role within the group is to carry out microarray analysis and so I wish to optimise the RNA isolation proceedure so that the isolation method is quicker and the RNA less contaminated.
We tried a few whole blood kits (Applied Biosystems MagMax whole blood kit and analytik-jena Innuprep whole blood kit), neither gave good results.
Ive tried using RNA later; took 3ml sheep blood into EDTA and then added directly to RNAlater. Tried isolating RNA using both column method and Applied Biosystems MagMax whole blood kit. Neither gave usable RNA. Someone told me that sheep cells are a different size to human/bovine cells so perhaps this may be a problem.
Ideally we want to isolate the RNA from both sheep and cow blood using a quick and cost effective method... any suggestions?
Posted 08 March 2009 - 03:42 PM
Posted 08 March 2009 - 06:25 PM
Add trizol, solubilize the cells/nuclei. Add chloroform. Spin.
Precipitate. At this point, I resuspend in water and then perform an extra acid phenol step to remove extra gDNA. If you do a DNAse step, that is fine too and the DNAse will be removed during the acid phenol step. It does add about 20 more minutes, but more reliable.
These steps only take an hour or so from start ot finish (even kit based), so it sounds like the rate-limiting step is the cell type fractionation, in which case the method used for RNA isolation is moot. As far as contamination goes and microarray analysis, the gDNA usually drops out depending on the method that is used to make the bait for the microarrays. For affy, they generally use a T7 based system wich can be conjugated to oligo dT primers, thus considerably reducing the affects of DNA.
Otherwise, that's pretty much the standard way.
Qiagen does have an RNAeasy kit, and I have had good success with it in the past, but the cost adds up if you are doing many samples.
Posted 07 April 2009 - 09:45 PM