Hi everyone.
I'm working with a fungus and I did the RNA extraction following the QIAGEN Mini Plant Kit and Trizol protocols, but all samples are contaminated with gDNA.
The samples were treated with DNase I and quantified in Qubit Fluorometer 3.0.
The DNA concentration was halved, but the gene was amplified in PCR rather than mRNA.
Do you have any tip?
Thanks in advance
genomic DNA is usually present in vast excess of RNA, so this isn't surprising. what kind of RNA are you trying to extract? what question are you asking that has you extracting RNA?
for example, if I am trying to identify an unknown RNA virus in a eukaryotic host, I would perform a trizol extraction (which doesn't separate RNA from DNA cleanly, as you discovered) and then do one or more steps to enrich for RNA (like DNAse treatment, which you found can be exhausted before the DNA content is significantly reduced) or specifically target the RNA - maybe reverse transcription, etc.
Hello Renan
You can try with mirVana kit isolation to RNA total. I work with it and all ok. I hace tested in RT2 profile Gene expression from qiagen and all the controls to gDNA were aceptabely negative.
Thank you very much, guys.
The extraction of total RNA is focusing on mRNA to heterologous expression (TOPO Vectors Invitrogen).
I guess the second treatment with DNase I can reduce significantly gDNA.
Try using a smaller sample per volume of Trizol and very carefully take only the top 1/2 - 2/3 of the supernatant. It's easy to pick up some of the gDNA in the interphase if you are too greedy. It also doesn't hurt to give it a longer spin during phase separation. And if you want to get fancy you could try a LiCl precipitation prior to DNase I digestion.
Agree with Patrick. Try taking upper part after trizol very carefully and if its a small amount of volume dont try to get more, risking g dna contamination. Its better to have little and pure rather than a lot but contaminated right? I used to take some rna from upper part, then re centrifuge for 10 mins and take more if I absolutely needed more.
If DNase treatment doesnt do the trick, try getting a primer that is specific to your mRNA but not your samples DNA.
Let us know how it went please. Good luck :)
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