Hi!
I am trying to understand whether it is important for me to use an RNA Cleanup kit (such as NEB Monarch #T2030S).
I am isolating microbial RNA from an environmental liquid sample, and I subject this RNA to subsequent DNAse I treatment. Does it really make a difference if I do not clean up the inactivated DNAse and buffer salts, and just go on with RT/qPCR? I am guessing it should work without a cleanup, but does such a cleanup help in some ways that I might be missing? What is your experience?
Thanks!
Artur
Without knowing the specifics, your PCR will likely work, but not optimally. You will carry over the salts from the DNAse buffer (assuming you used one), which may interfere with your polymerase. If you can do a cleanup (eg have enough RNA, and have the necessary supplies), I would suggest you do it.
Hello Artur Zaduryan
I agree with Péter Gyarmati . The most crucial step in RT-qPCR is sample isolation. No matter how good your assay design is, if the starting material is contaminated or degraded, you will not get accurate results. A good-quality sample is the starting block of good-quality data.
Digestion with DNase I removes traces of DNA and is compulsory if the RNA samples are destined for use in RT-qPCR. DNase digestion after the final RNA precipitation step involves adding extra salts and proteins to the sample and since this can affect the efficiency of the cDNA synthesis, additional purification steps are required. All traces of DNase and divalent cations from the reaction mixture have to be removed after DNA digestion is complete.
Best.
RNA cleanup kits, such as the NEB Monarch #T2030S, are typically used to purify and concentrate RNA samples prior to downstream applications such as reverse transcription (RT) and quantitative PCR (qPCR). These kits can help to remove contaminants such as DNA, salts, and proteins that may interfere with downstream reactions.
It is important to note that even after a DNase I treatment, DNA contaminants can still be present in your RNA sample. So, if you are performing downstream applications that are sensitive to DNA contamination, such as RT-qPCR, it is recommended to use a RNA cleanup kit to purify and concentrate your RNA sample.
RNA cleanup kits can help in several ways, including:
-Removing any remaining DNA contaminants after DNase I treatment -Removing salts and other contaminants that may interfere with downstream reactions -Concentrating the RNA sample to improve downstream sensitivity and specificity
In my experience, RNA cleanup kits are an important step in the purification and concentration of RNA samples, and can help to improve the sensitivity and specificity of downstream applications such as RT-qPCR. However, it's worth noting that some RNA purification methods are based on selective binding properties of the RNA molecules to specific silica beads, and this can lead to bias in the recovery of different RNA species. Therefore, it's always good to check the recovery rate of your target RNA species after purification.
To sum up, it is recommended to use an RNA cleanup kit for downstream applications of your RNA sample, particularly if you are performing RT-qPCR, to remove any remaining DNA contaminants, to remove salts and other contaminants, and to concentrate the RNA sample. However, it's worth checking the recovery rate of your target RNA species after purification.
Péter Gyarmati Hi Sir, If I have a very less concentration of RNA around 200ng/microliter of a total 10-15 microliter volume shall I go for DNase treatment before qRT PCR?
I am working on environmental chemical toxicant in a primary cell culture.
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