First guess - 2% is way too dense to resolve the two bands of rRNA on a small gel, especially if you are loading a lot of material. Which buffer system are you using? TBE/TAE or denaturing formaldehyde and SSC. Try running a small aliquot (100-200 ng) on a 0.8% TAE or TBE gel.

Also, do you get no amplification in a RT-neg control? Are your primers intron-spanning? i.e. is it possible that the single band is genomic DNA? Hopefully not!

If these suggestions are insufficient, please post more details - source of material, RNA purification method, picture of gel with size markers...

Good luck!

Hi Qurteeba,

I agree with Anna, maybe the % of agarose in the gel is too high. In our lab we routinely use 1% TAE gel.

Did you load a RNA Ladder in your gel? If so, the Ladder ran properly?

Do you know the amount of RNA that you have on your samples? Maybe you have low quantity. 

Good luck ;)

I don't think denaturing should make a huge difference to ribosomal bands, but it's definitely worth a try, as Anna suggests.

You don't need to use a denaturing gel (though it's optimal): you can just prepare your samples in loading buffer + formamide (aim for 60-75% formamide in final mix), heat to 65deg for 5 mins, cool on ice and load on a TAE/TBE agarose gel as you would DNA.

Other things to consider: the lower band (18S) stains with roughly half the intensity of the 28S, and often ends up hiding behind the bromophenol blue if you don't run the gel long enough (it's a long shot, but worth checking).

Thanks for the suggestions. I will try running the samples on a 1 % gel. The primers are designed from the exon junctions so I know the amplification is from RNA and not genomic DNA. Also the gel does not have any smear to show any degraded DNA or RNA.. will try the 1 % gel first.

Dear Qurteeba Qadri ,

It's better to increase run time or decrease % gel ( use from 1% gel).

respectfully,