Attached is my total RNA gel picture. Does anyone know that what is the fireball at the bottom of the gel? Is it symbolize degradation? or small RNA?
On the other hand, I did once DNase treatment on my RNA samples (DNase from QIAGEN, off column treatment) then precipitated with Ammonium acetate (left samples are total RNA from bacteria and the right samples are DNase-treated RNA samples shown in the gel picture). From the gel, we can obviously see that genomic DNA is eliminated from the total RNA. However, I obtained bands when I ran PCR to check DNA contamination. Anyone know what is actually happening?
My downstream work will be transcriptome sequencing.
Thank you so much.
Hello, :) its so cool you are doing transcriptomics
is degraded mRNA, its really degraded, some of your reactives its not ok, on the other hand you should do twice dnase step if you dont want any genomicDNA.
There is a lot of Genomic DNA in your gel, all of that white ghost above the first band is DNA, you cant say that there is not genomic DNA, there is in fact a lot. No DNA should be seen like an empty space between the top and the first band.
I strongly recomend that you first extract RNA without Dnase treatment, and see if degraded RNA is still present, if not then is the DNAse kit and you should buy a new one.
if there is still degraded, that would mean that is some step of your isolation, I always got degraded RNA with chemical methods, its really tricky to get good results, specially with those methods that demands >8 hours to get RNA.
I know its expensive but, buy a good rna extraction kit, the cost benefit worth it.
optionally, are you preparing all of the reactives with DEPC treated water? is your instrumental treated carefully to get rid of RNAse? degradation can happen even when you run the agarose gel... whats your method for all of this.
I would like to hear your reply about your methods, maybe there is a lack of treatment im some step.
best wishes and a lot of luck , RNA is tricky and I have suffered a lot to get good results too.
pd. there are rare cases where a fireball is created out of nothing, make sure you take a picture right after you put your gel over the transiluminator!!!!
Hi Gregory :) Thanks for your long reply. I am appreciate it a lot.
Here are some reply for your questions.
I extracted RNA from 25 mL of Serratia marcescens' overnight culture which has around OD 1.0 by using 1 mL Trizol. The concentration of RNA that I extracted is very high, around ~1000 ng/ uL. I did off-column DNase treatment for 15 minutes. Then followed by precipitation with 5M ammonium acetate and 100% ethanol and resuspended it with 30 uL nuclease free water. After that, I measured the concentration, A260/280 and A260/230 readings by nanodrop. Normally, I ran 1 uL sample and 1uL loading dye on 1% agarose gel with voltage 90 for 40 minutes.
For your information, I extracted RNA without DNase treatment and ran it on a gel, the results shown in the gel photo (4 lanes in the left) that I attached. There are also a fireball shown at the bottom of the gel.
There are few thought in my mind, I would like to share them with you.
1. If it is degraded mRNA, why we still able to see two intact rRNA bands in the gel photo? Is it because high intensity of the fireball at the bottom decreases the intensity of the rRNA bands when we viewed under gel?
2. My bacteria Serratia marcescens is actively expressing prodigiosin, a kind of secondary metabolite and it involves a gene cluster which contain 14 genes. Therefore, a lot of transcription and translation happens as a result there are many tRNA present in the total RNA and shown as fireball. Is it possible?
Thank you so much for the helps ;)
hello Siew :)
the first thing I should say is that the existence of luminiscence coming from above the rRNA bands in every lane of your gel means that only one DNAse treatment is not enough. Im used to perform more than one (usually 3) DNAse treatments to get pure RNA.
I dont see intact rRNA, a smearing below the second band implies some sort of degradation, or even gDNA partially digested.
I would tell you in my short experience that you should perform more DNAse treatments and maybe this artifact will be gone.
Hi Gregory :) thanks for your suggestion. I sent the samples for bioanalyzer to see its RIN. Unfortunately, i obtained very low RIN 3.5. The fireball (unusual band) might affects the bioanalyzer results as it might detect that as degarded RNA or a combination of small RNA and degarded RNA. But then again the intensity of the 23S and the 16S bands clearly remains very high. And also, on a Bioanalyzer this 5S band definitely does not look like degradation. The 23S/16S ratio also very high. Do you know what happen actually?
I am facing the same problem wherein I am getting a high concentration of 16S and 23S as well as fireball at the bottom. How did you solve this problem and any publication which supports the fireball is 5S and tRNA and not degraded RNA.
- Badges
- Science method