We are currently working on a project involving the extraction of DNA and RNA from various types of animal samples, such as whole blood, serum, faeces, etc. The objective is to detect various pathogens through Next-Generation Sequencing (NGS). Our approach for each animal is to combine all the extracted DNA and RNA (converted to cDNA) together (e.g., its DNA from faeces + RNA from faeces + RNA from serum + ...), thus reducing the number of samples to be processed during library preparation.
We have an extraction kit that allows us to either extract DNA and RNA together in a single tube, or extract DNA in one tube and RNA in a different tube. Since our intention is to mix them anyway, we are considering the former option. Nevertheless, we are uncertain whether this will impact the RNA-to-cDNA conversion. Will the presence of DNA affect the conversion process? Additionally, are there any potential effects on the integrity of the DNA? While extracting DNA and RNA together would offer significant benefits in terms of saving time, consumables, and reagents, we will not proceed with this option if it might adversely affect the quality of our extracted DNA or RNA.
Thank you very much for your time and assistance.
We use an enzyme called "reverse transcriptase" to create a complementary DNA (cDNA) sequence from the RNA fragment. This creates hybrid molecules that are a combination of RNA and cDNA
Are you sure this is the best approach?
"Detecting pathogens in mixed samples" is very much a needle/haystack problem, something PCR is basically perfect for.
NGS approaches are much more aligned with "we literally want to sequence EVERYTHING in our sample", which isn't really what you want, from the sounds of things.
You'll presumably have host DNA (and RNA), potentially in marked excess of your microorganism content, you'll have a ton of microorganism content that ISN'T pathogens, and you'll also have the fact that bacterial mRNAs are expressed differently to eukaryotic mRNAs (no polyA tails, so random priming is essential) which also means even if you successfully reverse transcribe anything, most of it will be ribosomes.
Also, mammalian genomic DNA tends to be HUGE, most of which is just repeats and low complexity stuff.
And most downstream pipelines are based around the assumption that you're either sequencing JUST gDNA or JUST cDNA, and trying to do both just seems incredibly counterproductive, especially when you're not actually interested in any of this, you just want to detect pathogens (which, again, you can do using regular cheap PCR).
I would...seriously have a think about your experimental plan before committing further resources to this.
We use the same approach for our animal research activities too. I can assure you that we have no problem generating cDNA from mixed RNA and DNA samples.
No, it does not affect the conversion, that’s why you use the reverse transcriptase enzyme which transcribes RNA into cDNA. Also, when isolating your RNA, make sure you carefully transfer the aqueous phase because it contains the RNA, and avoid transferring any of the organic layer/interphase. This helps to reduce contamination and increases the purity of the RNA. You don’t have to transfer all the aqueous phase. I hope this helps…
The presence of genomic DNA in the sample can be considered as a contaminant because of that during running Real time PCR, it affects quantity of cDNA (which is expected to come from expressed genes/transcripts). Since some DNA probes can bind to any double stranded DNA molecules, the presence of genomic DNA can affect quantification of cDNA.
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