Hi all,
I extracted RNA from bacteria and I run an agarose gel on these samples. How do I recognize if I have RNA and not DNA? I have three bands (in attachment) and I am not sure about what they are.
Thank you very much.
Hi ' It depend on extraction method you can distinguish between them
so according to protocole you used for extraction
regards
Hi Silvia,
check this out: https://www.researchgate.net/post/Checking_RNA_integrity_through_TAE_gel2
RNA looks more brighter than DNA on agarose gel as it is single stranded and EtBr has more ability to bind to it. The upper first band may be the genomic DNA contamination as gDNA is heaviest. the other two bands may be two forms of RNA as 28S rRNA or 18S rRNA.
Hi Silvia,
I believe if you used an RNA extraction method then you should have RNA bands! you can check your sample if you still have any for their purity by nanodrop for example, or what is the RNA size you are expecting? I mean see that bacteria's DNA band size and then compare with what you have.
all the best
You can use a Dnase in your protocol to remove DNA from your RNA extract, also you can use UV spec to determine the A260/280 ratio to determine DNA/RNA content. A ratio of 2 is widely considered pure RNA.
The RNA stands out as thick bulbs while the DNA remains as light bands and much thinner.Also RNA always moves first and DNA follows
Use this protocol u will get ood results.
First make pellet from 2 ml O/n grown culture and incubate for 1 hr in 10mg/ml lysozyme. after that add 1 ml Trizol and vortex. note that mixture become little transparent . then add 200ul chloroform mix well n incubate on ice for 15 min, centrifuge at 12000xg for 15 min at 4C. Then transfer the upper 150ul aqueous phase to new 1.5 ml tube and precipitate RNA by gentle mixing with 0.5 ml isopropanol, incubate on ice for 20 min, centrifuge at 12000g for 10 min at 4C. discard supernatant wash RNA pellet 2 times with 75% ethanol and then dissolved in 20ul Nuclease free water. Now u will get good Result after that Treat ur RNA with DNase.
Hi Silvia,
Coming straight to your query, it seems RNA is degraded in your sample and the top band may be DNA. RNA generally shows two consecutive sharp and clear 28S and 18S bands in 2:1 ratio. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp bands (as observed in your sample). Completely degraded RNA will appear as a very low molecular weight smear. Generally DNA travels less as compared to RNA in same sample (in case of DNA contamination) If you are getting a band on top along with RNA bands, that may be your DNA contamination and should be treated with DNase I. I am attaching a gel image which may help you to recognize the bands in future. Hope this may help you. Please do not hesitate to ask any further questions.
All the Best
they are orderly top to bottom, genomic DNA, 23s rRNA and 16s rRNA.
Your RNA is not really degredated. Your agarose gel is poor quality, you have same band quality in the marker region too. I suggest that you to prepare agarose gels immediatly after you melted them, without letting it cold outside of the cassettes. Then you will see better quality gels.
Hi Silvia,
from the view of the DNA ladder , it seems that you did not allowed the gel to completely harden before taking off the combs and this had distorted the well shape and hence the shape of bands. Also you should be accurate in reducing the melted agarose temperature to about 60C before mixing it with EtBr as this will give you some haziness in the gel view. To improve this,try to photograph the gel in a back to bottom way or upside down and rephotograph it you will notice a clearer and sharper band appearance and also reduce your run time. DNA is obviously still present in your gel if you treat your sample with DNase ,this might further degrade your RNA ,and not all downstream application necessitate the removal of DNA from your RNA sample.
Hi Ali,
As you already described that rRNA is abundant (approx 90%). As per my knowledge mRNA is hard to detect on agarose gel as it is less then 5% (mostly 1-2%) of total RNA.
Dear Silvia, First of all I want to ask you about your ladder, what was your ladder? Regarding the three bands, did you used DNAse? If not. It possible that there is plasmid DNA in your sample which might migrate in three bands.
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