Low RNA yield after extraction by RNA blood mini kit. - (Dec/21/2009 )

Hi,
I have stuck in a process of RNA isolation from blood.

I would like to extract the RNA from rare cells in peripheral blood that should be enriched before extraction step right? Method that I've used is density gradient centrifugation by using Histopaque 1077. I started from 10 mL blood sample, followed by the Histopaque's instruction and finally I got large amount of cell pellete. Then I extracted RNA by QIAamp RNA blood mini kit. While I was adding the RLT buffer, the mixture was very viscous and many foam occurred. According to the instruction, additional RLT buffer is recommended and need another column. I decided to use one column and didn't add any RLT buffer then transfer to the spin column. So, I noticed that the volume of the lysate (after centrifuged) was lesser than the volume of added RLT buffer. Then I checked for RNA concentration by nanodrop spectrophotometer and the result show the low yield of RNA when comparing RNA that was isolated from 1mL of whole blood.

What should I do? Any suggestion?

Thank you

usually column based kits from Qiagen give low yields, that's for sure, although the purity is high.

I extract RNA with Qiagen's RNA Mini from cells and it always gives me lower yield compared to Trizol method.

Curtis on Dec 22 2009, 11:45 AM said:

That's true about Qiagen's kit gives low yield but its column capacity is not full yet. According to manual, they claim that the column capacity is 100 microgram but I got RNA concentration just around 8-30 microgram. It should be more than that.