In addition to the points raised by David on buffer conc and other factors, I suggest you to first be sure about your RNA extracts. In the absence of marker, The bands look like only small RNAs to me. So be sure that your RNA extraction method and your lab is RNase free. While running RNA in formaldehyde gels you need a special RNA loading dye, which includes denaturants. Try to follow a standard protocol.

Hi Kamelia, numerous factors here may play some role in this entire scenario you have just stated.

You did not mention the source of your RNA (FFPE, fresh tissues, snap-frozen tissues, microbial, cell lines, blood, plant). It can help to think about the involvement of any other possible flaw beyond the gel level.

But, if your RNA is good in quality and quantity, absorbance ratios are perfectly fine then you are good to go for the run. So use always a dedicated gel tank for RNA gels or wash it properly with DEPC-treated water or bleach and dry well.

Also, did you already perform any DNAse treatment on your RNA samples?

gDNA removal phase often destroys much of the RNA you have, if it is not performed properly like using improper concentration of EDTA or any other DNAse inactivation reagent, which is used after the incubation time of gDNA-removal assay to destroy the leftover DNAse. Since this phase requires heating of the RNA sample so an instant chance of divalent cations-dependent RNA degradation is always there in the absence of any divalent cations quenchers (EDTA).

You must run your gel with an appropriate RNA ladder and RNA loading dyes, which must be heated first (70 C for 10 min or as per manufacturer) and then chilled on ice prior to well loadings.

Usually in denatured RNA gel we load above 2 ug of RNA and maximum being 5 ug, proper run time is 60-65V for 45-50 minutes, or 2 - 4 hours at 55 C.

Here 5V / cm means if your gel, for example is 10 cm long, so you must apply at least 50V current (5 * 10). Remember high voltages badly affect bands' resolution, especially be careful when your sample is RNA.

5 - 10 x MOPS when used it ultimately becomes 1x upon diluting with all other components of agarose preparation. MOPS buffer pH should be checked as RNA is highly unstable below 2 pH and at or above 9 pH. MOPS plus formaldehyde mixture is kept warm at 55 C in water bath and is poured onto melted agarose while warm.

Post stain with SybrSafe. If you have UV illuminator so you must be aware that prolong exposure also damages RNA and never ever reuse SybrSafe solution, as this diminishes its sensitivity.

Regards...

David Farringdon Spencer

I make the gel using 5x MOP buffer but I use 1x running buffer. On this particular photo I didn't run the ladder as I just wanted to test the gel itself.

My samples are RNA extracted from snap frozen tissue.

I add syberSafe+37% formaldehyde to the gel when cool down (55 degrees) then pour to solidify.

I prepare the samples using 2ug RNA and sample loading buffer (ratio;1:2), then add the RNA loading buffer, heating up at 60 degrees for 10 minutes, then on ice for 2 minutes.

The voltage is still my problem as the second time I ran the samples I did 90V over 90minutes, because samples were moving very slow along the gel, recommended is 2/3 of the gel.

So Jawwad Ahmad You are right I think high voltage over long time destroyed my samples and I got a photo similar to degraded RNA.

So this time I will use 65V for 45 minutes.

You can use 1% TBE gels (no formaldehyde necessary) to run RNA gels. The TBE will remain cool and not heat up significantly. It does look like there are 2 bands in your gel image, corresponding to the larger and smaller rRNA subunits, but the gel needs to be run for longer to separate the two bands. The ratio of intensity of the two bands can be used to asses quality. (ie: more of the smaller band than the larger means your rRNA degraded and produced the smaller band, you ideally want 2x as intense a band of the larger than the smaller band).

Best of luck!

David Farringdon Spencer

"RNA work in the 70s"