Our team is looking for viral RNA in the environment. We took swabs off of doorknobs using cotton dipped in water and then submerging that swab in viral transport media. We also wiped 1 surface with disinfectant spray thoroughly as a negative control and took a swab of that. We did RNA extraction of 6 environmental samples (including negative control), and 5 samples of our model virus (Eliat) in our transport media, as well as in water, with and without our cotton swab.
Every single sample showed a decent curve on the nanodrop at 260 after the RNA extraction.
Every sample showed a concentration of around 80 nanograms/microliter.
Every sample showed a 260/280 of around 3.40
Every sample showed a 260/230 of 0.17 - 1.02
It seems so strange to me to see these results be so perfect.
My questions:
Could this be contamination in all my samples?
Could the kit I used be saturated in RNA which is why I found the same concentration?
I'll double check the disinfectant I used but is it possible that it didn't destroy RNA on the surface I applied it to?
What I'm going to proceed doing:
I have primers for Covid (what we were testing the environment for) as well as our model virus however the model virus has no control for PCR. I plan on running an RT-PCR tomorrow but I highly doubt I'll find covid. My model virus primers have yet to be used so I'm curious if they even work. If they don't work I have no way to verify if my RNA extraction was successful or not.
Any suggestions?
your od260/280 looks high. 1.8 would be very good but 3.4 is probably reagent contamination due to incomplete washing of the column. Is it possible that your positive pcr values come from post pcr dna contamination re amplifying? Are you running a no template control sample (water) in your pcr which has not been through the purification process and is this sample showing negative after pcr?
On top of Pauls answer, is the cotton and water from a sterile source (no template control will help answer this) and are you touching the cotton at all with gloves or hands or is it afixed in a swab format as it could be contamination from that source too. Same applied for all tubes etc
Also it depends on the desinfectant you're using, as it's possible in case you're using just alcohol that you're precipitating the RNA, not completely degrading it. In addition, you're risking DNA contamination in general with the RNeasy kit, I'm usually purify with DNase I after the RNA extraction. Nanodrop is not preferred for RNA quantification, better to use fluorochrome-based technique as done with Qubit.
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