Hi there,
I am extracting RNA from environmental samples like soil. I have a three stage protocol. First I use a Chloroform/Phenol based extraction according to Toewe et all, then I am using a Macherey and Nagel Kit and at the end I am using HighPrep Magnetic Beads from Biozyme.
When I measure my sample afterwards on the Nanodrop I get something between 350-800 ng/uL RNA with a 260/280 ratio of 1.93-1.99 and a 260/230 ratio of 1.99-2.18. When I measure the samples on the Qubit I get similar concentrations and if I load the samples onto the Bioanalyzer then I only get one big Peak (not two), so I thought I still might have DNA left in my samples and for further downstresm processing I decided to do a DNA removal.
Therefore, I used the Turbo DNA free Kit, however, after using the kit the quality of my RNA samples dropped according to NanoDrop to 260/280 1.70-1.73 and 260/230 1.80-1.90.
Does anyone of you has an idea why the quality of my RNA decreases after DNA removal and what I could do?
Thank you!
Dear Eileen, your first extractions some DNA contamination can present. After removel of DNA the absorbance values can decrease. In your first measurements absorbance of total nucleic acids are measured. If you plan to use in expression studies and cDNA synthesis it can work with 1.7 ratio.
Best regards.
I think also the gel Picture looks really weird, or what do you think?
Sample 1-3 (left) before DNA removal via Turbo DNA-free Kit and samples on the right the same samples after DNA removal via Turbo DNA-free Kit.
Sample 1 was only extracted via CTAB and Phenol/Chloroform according to Toewe, Sample 2 was extracted like samples 1 + Macherey and Nagel clean-up Kit and Samples 3 was extracted like sample 1+2 + Bagnetic Beads.
It looks like what you got there is not RNA at all or a very degraded sample. Even when looking at a gel with RNA coming from different species you should see the rRNAs as very sharp bands (see attachment).
I'm also concerned about the nature of your gel, was it a denaturing gel? How did you store your RNA before loading the gel? RNA is easily degraded if not stored at -80 ºC.
Although the ladder looks great I cannot see anything resembling an RNA sample in that gel. Maybe the problem is not with your RNA but with the gel you prepared.
http://production-oms.s3.amazonaws.com/static/Image/Lonza%20Quantitative%20RNA%20Fig%202FlashGel.jpg
Hi Ander,
yes, I thought that it looks really not great at all too. It's a normal 2% agarose gel and yes, we store the RNA at -80ºC and we measured on NanoDrop, Qubit and Bioanalyzer. I attached the Bioanalyzer results for one of the samples, before and after DNA removal.
Hello again, Eileen,
I was also expecting a two-peak pattern in the bioanalyzer, something's really wrong there. I guess DNA treatment may be the cause, but if you carefully followed the protocol then I have no clue about what would be the cause of the drop in RNA quality, sorry.
My only suggestion now would be to change the RNA extraction protocol and try TRIZOL, I've never had any problems when using it and my samples are almost free of DNA even without treatment.
Hope someone else can help you with the issue, good luck!!
Hi there,
I tried the RNA extraction again and changed a view things and now I've got this (look attached picture). Do you think that's okay and really RNA? Just seems to good to be true.
Well, well, what do we have there!! That's a perfect RNA extraction indeed, your RNA integrity looks good and no gDNA is visible. Sometimes it takes time to optimize protocols and it looks like you finally got it, my congrats, Eileen.
- Badges
- Science method