Hi, I am currently trying to isolate RNA from whole blood. I have frozen blood samples - preserved in RNAlater stored at - 80. Samples were collected in RNA later (300 ul blood sample and 1.3 ml of RNA later) and immediately frozen at -80ºC. These samples were stored at approx. 6 months at -80. Then, I tried isolating RNA with TRI BD reagent (sigma), and also through the organic Trizol method, but not succeed with any of them. In the agarose gel, I did not observe any RNA traces (18S and 28S rRNA subunits nor the smear of degraded RNA). RNA seemed to be totally absent. Can anyone provide me with a protocol for the removal of RNAlater from the samples - thawing, centrifugation, etc?
Thanks.
Pragati Raghuwanshi Most probably your sample is destroyed. RNALater is not recommended to preserve white blood cells in whole blood. To store white blood cells, you should isolate them from whole blood and resuspend in RNALater.
While working with blood samples, I will suggest always separating plasma, isolating RNA, and then storing them.
Try the following tweak.
*Incubate at room temperature after addition of chloroform and invert carefully until you observe color change.
*Take only 70% percent of the aqueous Phase during separation after addition of Chloroform
*Use isopropanol for precipitation
* Precipitate overnight at -20 degrees Celsius
Depends on what you want to achieve, but typically you cant recover high quality RNA from frozen blood no matter what buffer you use
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