Hi Elham,

If your marker is not there, then your problem is clearly at the electrophoresis stage or earlier and there could be a handful of reasons. Let's suppose that the RNA handling is fine then in order to rule out some possibility let's start from the obvious ones:

Let's leave the ladder issue for the moment.

2 ug vRNA should be more than enough RNA to visualise on a gel unless degraded. How big if your vRNA? and what's the % of your agarose gel? You surely have consider this before but big vRNA may not enter the gel if your gel % is too high.Either way, I don't think this would be the problem since most vRNA are quite small unless this is an special one. Also, retention in the wells could be simply some contamination or high salts in your preparations.

What voltage are you running your RNA at and for how long? Perhaps you've lost them.

How much formaldehyde are you using in your gel/buffer? which buffer? Does your loading buffer contain formamide or formaldehyde or both?

VERY IMPORTANT: check the pH of ALL your buffers...neutral pH should be used for RNA as this is very sensitive to alkaline pH, so the temperature generated during the run (or heating pre loading) plus alkaline pH will definitely destroy your RNAs.

For now, I'd suggest to run a denaturing (non-denaturing works ok but denaturing is better) eukaryote total RNA. Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact and if you process them in parallel it could give you a better idea on how your handling is going.

Good luck!

Is your EtBr in your gel or in your sample? For formaldyhyde gels you will want the stain in your sample only it is too hard to stain the entire gel because the EtBr is everywhere and the background is too high to see any bands very clearly. Sometimes you can destain overnight in buffer to get rid of the background, but the best way to see your bands is to only have EtBr in your sample buffer. Hope that helps.

Thank you for your advices,

My RNA size is about 1,7 kb which I run it on 1% formaldehyde gel. I don’t think that the RNA has high concentration of salts since I use QIAGEN kit for its extraction.

To run the RNA, I start with 100V and after my samples run out of the well I reduce the voltage to 80 which takes approximately 1,5 hour to pass 2/3 of the gel.

In a total volume of my gel which is 80ml, I use 12ml Formalin and as a running buffer 1x MOPS.

I also checked the pH of my 10x MOPS which was around 5 but not more than 7. I adjusted then the pH to 7 to see if it makes any changes for next run.

For now, I have extracted total RNA from the hepatocytes to check robisomal RNA on the gel as Luciano has suggested and I will also stain my RNA by adding EtBr directly to my loading buffer.

lets see if these work, I'll focus on my gel to find out the problem.

Hi Elham, good to hear your on top of things! For what you mention in your text everything seems to be perfectly done. Let me know how your ribosomal RNA looks like and we can keep thinking about possible causes from there. Finger crossed!

Sounds good, good luck and let us know how it works out for you!

Hi again :)

I run my RNAs on normal Agarose gel as well as on Formaldehyde gel.

with 2.5 ug of RNA mixed with RNA loading buffer and EtBr prior to loading (without boiling at 65'C) on 1% Agarose gel I got two clear ribosomal bands. While running about 5 ug RNA mixed with loading buffer and EtBr, boiled for 5 min at 65'C on 1% Formaldehyde gel gave very weak bands with smear!

I'm not sure what will happen if I use normal Agarose gel to run my RNA and I'm wondering why my formaldehyde gel doesn't work well!

Is your ladder prepared in the same loading buffer and how does it look on the gel? Is it also smeary? Without seeing the gel it is hard to determine if the RNA is degraded or if the something about the gel or running buffer is causing the problem. From what you describe it could be either your RNA is degraded in the heating and loading steps or that your gel and buffer compatibility might be a little off. Did you run the viral RNA alongside the total RNA and is there a clear band or a smear? That should be a strong indication of degradation as well.