I am using a Qiagen AllPrep Kit to extract nucleic acid and protein from PBMCs. My RNA concentrations are consistently low and of poor quality (260/280 ratio < 2 and concentrations below 40 ng/µL).

UV-Vis measurements are highly variable when taking measurements on the same sample - today quantities ranged from 0 to 4 ng/µL. I ran a denaturing agarose gel on a good quality standard RNA sample lent to me from another lab of the same concentration as my extracted RNA. I saw a band for the standard but not for the sample I extracted. I am now thinking there may be no RNA in my sample to begin with or it is of an extremely low concentration.

I am using the appropriate amount of lysis buffer for my cell quantity, the correct ethanol (anhydrous, >96%) and am even using extra steps suggested on the Qiagen website (spinning to get rid of excess buffer RPE and letting nuclease free water incubate on the spin column membrane for 10 minutes before eluding the RNA).

Lab mates of mine have suggested using TRIzol instead of a spin column system. Any advice and/or ideas would be greatly appreciated. Thank you.