QuickPrep Total RNA Extraction Kit From GE Healthcare

This kit designed for rapid isolation of total RNA from different types of tissue and cultured cells. It is based on a procedure that utilizes the disruptive properties of guanidinium thiocyanate (GTC), followed by lithium chloride (LiCl) centrifugation, which preferentially precipitates RNA, and then washing using cesium trifluoroacetate (CsTFA). This type of RNA extraction is environmentally friendly, does not require phenol or other hazardous solutions and therefore, can be performed very conveniently on the lab bench without the need for a fume hood. Tissue or cell disruption is done in the GTC buffer; this helps to repress endogenous RNases and dissociate cell components from RNA. At this point, RNA extraction can proceed according manual instruction or the disrupted cells can be stored in the GTC buffer at -20ºC or -70ºC, which adds to kit's flexibility. Another appealing point is the possibility of using this kit for total RNA extraction from different amounts and different kinds of cells and tissues.

I used this kit very successfully for at least three years during my PhD studies, isolating total RNA from as little as five Xenopus embryos. The resulting total RNA didn't show any genomic DNA contamination, tested by PCR using primers for several genes, and it showed good quality too, tested by agarose gel electrophoresis.

Now, in my new laboratory, when I started a new project which involved a lot of RNA isolations from different kinds of tissues and cells, I went to use this familiar kit. I tried to isolate total RNA from several amounts of cells, starting from 2.5 x 10⁶ isolated PBMCs (peripheral blood monocytes) up to 2 x 10⁷ PBMCs. These cells numbers are well above the minimum quantities mentioned in the kit's manual which claims that it is possible to purify high quality RNA from samples ranging in size from 25 mg to 1 g of tissue or from 10⁶ to 10⁸ cells. Unfortunately, I didn't succeed at all. I didn't get any total RNA. In parallel to RNA extraction using the QuickPrep™ Total RNA Kit, I performed total RNA extraction from the same number of PBMCs using a traditional phenol/chloroform method; I obtained 4 to 10 ug total RNA. I contacted GE Healthcare Technical Support for help, but it was an unsatisfactory experience because they simply told me that this kit isn't intended for such cell amounts and recommended that I extract from 10⁸ cells per sample.. Also, our neighbor laboratory didn't succeed in total RNA isolation from 50 mg of mouse aorta using this kit.