Hi
i did RNA extraction following promega kit, i got highe yield of RNA but i found DNA in the samples. any idea what can i do? The kit steps include a DNase treatment but it seems the digestion is not working. Any suggestions would be appreciated. :)
Hi Faisal,
just do another DNase treatment with your purified RNA. Always keep in mind that DNaseI is a very sensible enzyme, so its activity suffers severely by wrong handling, storage conditions or even vortexing (only mix by pipetting carefully up and down 3-4 times). Sometimes it is worthwile to prolong the DNase treatment for an hour. DNaseI from different companies also might have a very different performance.
Hope this is helpful and all the best for your experiment!
Gertrud
Hello Faisal,
you can try the DNase from Qiagen, however since DNase is highly sensitive, its treatment in RNA sample sometimes lead to non-specific RNA degradation. This happens when only a small quantity of DNA is present in the sample and the enzyme gets only RNA to act on. Also, if you could upload the gel image of your RNA sample, I may help you better as sometimes a single band appears above the 28S RNA band which is hnRNA, not the DNA. DNA because of its high mol weight usually appears near the loading wells.
Thank you Gertrud and Chitra,
DNA has been detected by bioanalyser. so I am sure there is DNA. I will perform DNase again from different kit and see how it goas
Dnase from different vendor, even different batches from the same vendor may have huge varation of activities, so try another brand (i.e RQ1 DNase from Promega).
Another suggestion is,use a RNA extraction kit that has DNA removal column, which will get rid of at least 90% of the genomic DNA contaminant.
Hello Faisal,
As correctly said by Chitra and Gertrud, you try the RNA Isolation by using DNase from Qiagen or Promega also follow the temperature conditions exactly what is recommended in protocol.
Alternatively you can use RNA extraction kit this will remove most DNA.
The Promega kit should also include the DNase treatment so you should not have any residual DNA in your RNA yield, may be you skipped that step or was not effective. If you actually quantify DNA from your RNA yield then you may try an additional DNase treatment again. Good luck!
Hi Faisal
I´m trying to isolate pure mRNA from a M. tuberculosis using a Promega Kit (SV Total RNA Isolation Kit) and I´m having the same problem as you: DNA contamination. Did you solve your problem?
I hope that you have solved your problem. Alternatively, I will just point out, that we have oligos that can be designed to specifically bind to DNA. You can attach such oligos on a bead and remove the DNA impurity, or you can block binding of primers to the DNA, only seeing your RNA in amplification reactions. The advantage of this approach is that you do not degrade your RNA (which is sometimes seen when using DNAses). If you need further input please contact us at [email protected]
Hello everyone,
I have somewhat the same question. I extract RNA by pBiozol method for miRNAs (similar to Trizol), which i think is a very ''cheap'' protocol because of many flaws done by the manufacturer (but i have no other way because my lab is ''poor'').
Anyways, after mastering it for about 50 times, I usually can get about 1k to 10k ng of RNA from it.
After that, i tried to apply DNAseI protocol, but that removes everything.... No RNA, no gDNA, nothing. My professor told me to add few steps of Trizol protocol after the incubation period (in DNAseI protocol). Results were the same.
, which doesn't make any sense, but i have to do it for 1000s of times more, because among stupids and misers what else can i do?
I personally believe that the amount of RNA and DNAseI used must have some relation.
Experts, please i need your valuable tips, so that i graduate from this hell
The very best method for this is the use of kit
follow:
https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0013178_Removal_Genomic_DNA_from_RNA_Preparations_UG.pdf