Using RNA minikit for DNA extraction - (Jan/10/2017 )
Need to start a study which aims at looking at RNA and DNA viruses plus bacterial DNA in cerebro spinal fluids. Have no funding. There is a left over Qiagen RNA mini kit. Any one has any experience of using this do detect both DNA and RNA? Any suggested hacks to improve DNA yield? e.g - modification of carrier RNA content.
Am thinking of using it without using DNAse.
Thank you in advance for the answers.
which minikit is it? viral? blood?
the literature that comes with the kit (or which you can download from qiagen's website) will tell you if you can also isolate dna with it.
since you have no funding and can't purchase kits, you may have to go old school and use the procedures which led to the kits (see sambrook).
Qiagen cites differences between DNA and RNA colums, the RNA ones modified to preferentially not bind DNA (but they do), they also look different. But it's proprietary. In any case buffers are different.
I was now looking at QIAGEN products and they have a kit for copurification of viral RNA and DNA, obviously colums named QIAGEN Mini columns and QIAamp MinElute Columns do bind both. If you have such named columns in your kit, they are likely the same and you can look at buffer differences, or find resources, some QIAGEN buffers have been "decoded" and some of the recipes are available.
The kit you should look up is QIAamp MinElute Virus Spin Kit, you can dowload the handbook. This obviously uses the same MinElute columns as for DNA, but I never found a RNA kit using those. But if only columns are the issue, you may be able to find someone who has any MinElute columns and can maybe exchange with you for some MinElute ones.
Hi there, sorry for the delay in getting back. The kit we use is the QIAMP Viral RNA mini kit. The product literature states that both DNA and RNA will be purified together. I am guessing of we use the kit both would be extracted.
What I need to decide is if to use the carrier RNA or not.
The next issue is, if one designs a multiplex PCR that looks for both DNA and RNA based organisms together, that is the template to use. Convert the RNA extract to cDNA and then use that maybe?
Using reverse transcriptase will convert RNA to RNA-cDNA hybrids and leave the DNA there alone. Then the cDNA and gDNA from viruses can be amplified together. However, as in case of other RT reactions, resulting mix has to be used to maximum of 10 % of PCR reaction, so if you have low concentration of nucleic acids, this may be a problem, more RT mixture can inhibit PCR, you need to test your multiplex reactions in this conditions.
But, since it is a virus RNA and not transcripts, the RT step had to be carefully designed. Depends a lot on your kinds of viruses you want to detect. Usually for mRNA olido dT primers are used in RT step, that bind to polyA tail. Not all RNA viruses do have polyA tails however. Other primers for mRNA is random hexamer, which is used for viruses or third option is to make RT with the virus specific primers. But if it's a ssRNA virus, you need to design it in the correct orientation. Without purification though, virus specific primers may interfere later in PCR step.