Isolated RNA from the following samples stored in RNAlater:
- TH52 3.28.14 c-fluid
- TH54 3.28.14 c-fluid
- CH55 3.28.14 c-fluid
- CH56 3.28.14 c-fluid
- CH57 3.28.14 c-fluid
- TH65 3.28.14 c-fluid
- TH66 3.28.14 c-fluid
- TH67 3.28.14 c-fluid
Spun samples 5000g, 20mins @ RT to pellet any cells. Discarded supe. Resuspended cells/debris in 1mL TriReagent. Disrupted cells by pipetting and vortexting. RNA was isolated using the Direct-zol RNA Miniprep Kit (ZymoResearch). RNA was DNase treated on-column, as described in the manufacturer’s protocol, using DNase I. RNA was eluted from the columns using 25uL of nuclease-free H2O and spec’d on a NanoDrop1000.
So, this is disheartening. Overall, the RNA looks pretty crappy; poor 260/280 ratios and a general shift in absorbance to 270nm. Plus, the yields aren’t that great. Maybe RNA left on the column and/or some sort of contaminant pushing these readings out of whack?
I will perform another elution on the columns with 50uL of nuclease-free H2O and spec that elution set:
There’s still a shift in the peak absorbance in most samples to 270nm… I’m going to combine the two sets of elutions and spec:
Although the 260/280 values are significantly better, there’s still this persistent shift of peak absorbance to 270nm. I contacted technical support for the kit and they say the absorbance shift is indicative of phenol contamination. They have advised that I add a volume of TriReagent to the RNA and re-run it through a new set of columns, following the entire RNA isolation protocol.