What species are you looking at? There are big differences between vertebrate and invertebrate rRNA. See the quote belwo from a protocol from:

http://primerdigital.com/rna.html

"The rule for vertebrate rRNA - that in intact total RNA the upper (28S) rRNA band should be twice as intense as the lower (18S) band - does not apply to invertebrates. The overwhelming majority have 28S rRNA with a so-called "hidden break". It is actually a true break right in the middle of the 28S rRNA molecule, which is called hidden because under non-denaturing conditions the rRNA molecule is held in one piece by the hydrogen bonding between its secondary structure elements. The two halves, should they separate, are each equivalent in electrophoretic mobility to 18S rRNA. In some organisms the interaction between the halves is rather weak, so the total RNA preparation exhibits a single 18S-like rRNA band even on non-denaturing gel. In others the 28S rRNA is more robust, so it is still visible as a second band, but it rarely has twice the intensity of the lower one."

It's possible that you only thus have the 18S band and it's being called incorrectly, have you run it on a gel with a DNA ladder to verify the size?

What species are you looking at? There are big differences between vertebrate and invertebrate rRNA. See the quote belwo from a protocol from:

http://primerdigital.com/rna.html

"The rule for vertebrate rRNA - that in intact total RNA the upper (28S) rRNA band should be twice as intense as the lower (18S) band - does not apply to invertebrates. The overwhelming majority have 28S rRNA with a so-called "hidden break". It is actually a true break right in the middle of the 28S rRNA molecule, which is called hidden because under non-denaturing conditions the rRNA molecule is held in one piece by the hydrogen bonding between its secondary structure elements. The two halves, should they separate, are each equivalent in electrophoretic mobility to 18S rRNA. In some organisms the interaction between the halves is rather weak, so the total RNA preparation exhibits a single 18S-like rRNA band even on non-denaturing gel. In others the 28S rRNA is more robust, so it is still visible as a second band, but it rarely has twice the intensity of the lower one."

It's possible that you only thus have the 18S band and it's being called incorrectly, have you run it on a gel with a DNA ladder to verify the size?

It could have degraded during the extraction or as mentioned by Michael, the intensity is low for it to be visualized.

Concentration of 28S RNA is usually twice of that of 18S, thus in low concentrations only 28S band is visual. Maybe you should increase the amount of RNA that you are loading. Keep in mind that 28S rRNA has longer half life than 18S, use fresh samples and try to decrease your extraction time.

You can also visit this webpage:

http://www.lifetechnologies.com/ir/en/home/references/ambion-tech-support/rna-isolation/tech-notes/assessing-rna-quality.html

As long as you don't have smear along the gel, your RNA is fine.

Micheal parsons, My organism is brown algae Sargassum.

@Lekha Margassery I could think it as degradation but 28S degrades faster than 18S. opposite in my case.

@ Nikolay Dimov I am saying it as 28S becasue in bioanalyser it is coming at 45 sec. and there are very few 2-3 peaks at 38-39 Sec.

I would recommend a denaturing agarose gel. It is also worth having a look at this publication "Journal of Applied Phycology, October 2013, Volume 25, Issue 5, pp 1277-1285"

I'm working with bacteria, Acetobacter species, where the 16S rRNA appears to be split in two. It was the same for other cyanobacteria and enterobacteria.

Maybe in your case it happen something similar. But I agree with the fact that as long as you do not observe a smear, the preparation should be fine.

Eman El-Abd I am following that paper because that is the most suitable paper for Sargassum but my results are not very much similar to them...

@ Barro, I also think it should be fine as there are not smear in the band.

I run the gel with the manual extraction as I was doing before but before RNease MIni elute kit purification and I get 2 bands. There must be something wrong in my kit.

I agree with all recommendations, especially with Eman El-Abd,own. The denaturing agarose gel will give you a good separation between bands. But if you are unsecure , I recommend to take your samples to Agilent Bioanalyzer and to check you sample quality and the 28S/18S presence.

Good Luck

Yes, you should check gel on formamide-MOPS based denaturing gel. This would definitely give you good picture.

Is it vertebrate or invertebrate (e.g. insect) total RNA preparation? Are you sure that what the single band migrates as 28S rRNA rather than 18S? Note that in insects most of 28S is naturally cut approx in the middle and both parts of 28S migrate together with 18S showing single band on the gel and single peak on Bionalyser.

Good morning,

I recommend you read the following paper: Winnebeck EC, Millar CD, Warman GR. 2010. Why does insect RNA look degraded? Journal of Insect Science 10:159,

available online: insectscience.org/10.159. It can help you.

Good luck

Two questions

1) How are you disrupting the tissues/cells.

2) What method or kit are u using......I see a trend here......

I am pulverizing tissues in liquid nitrogen in mortor pestle followed by 2 min vortox and then incubation at 65 for 10 min.

I am using CTAB extraction buffer and purifying the RNA with Qiagen kit. Previously I was using mini elute kit and now I am using Qiagen Plant MIni RNease kit.

May I know what trend you are talking about.? Nobel Egekwu

Thanks..

What exactly were your conc...and 260/280 ratio?....

Concentration is in the range of 250-350 ng/ microliter and 260/280 ratio is around 2.00- 2.05.

Hi Amit,

From these reading it seems that you are getting high quality RNA yield. In higher order animals (for example mammals), it should appear two prominent bands corresponding to 28S and 18S rRNAs on gel.

What type of gel electrophoresis method you are using for checking RNA integrity? denaturing or non denaturing? at what position your getting single band on gel?

As previously mentioned by Eugene Ryabov and Jose Manuel Estivalis, there may be hidden break mechanism occurring in your 28S rRNA species from your sample. Refer this article: Ishikawa H. (1977) Evolution of ribosomal RNA. Comp. Biochem. Physiol. B 58, 1-7.

Regards,

Govindraj Chavan

On a standard agarose gel, one must observe intact 28S and 18S (rRNA) bands and sometimes 5.8S rRNA also. They are abundant and will appear prominent. The mRNA will appear as a very thin smear spread along the entire lane, which is hard to see most of the times.

Respected all. I am using RNaesy kit for RNA isolation from bacteria I am also getting one band on non-denature gel electrophoresis. 260/280=1.99 to 2.1.Concentration 54.55ng/micro litre. What should be possible redone for 23sRNA and 16sRNA?

Try the denaturing gel, slow run, and fast UV visualization. If you still see one band that is definitely degradation.

Hi Amit 

Hope you have been able to troubleshoot the problem. I am also facing the same problem with my thyroid cancer tissues.  I am getting on;ly one bans corresponding to the 18 S rRNA position. Hope you can help.

hi Qurteeba,

Sorry to hear that you are facing the same problem. For me the problem was existing Qiagen RNase kit. I changed to the RNase plant kit Qiagen and it worked well.

I do not think my solution will help you much as you are working on completely different tissues. 

Hi Amit,

I've prepared 4 samples and one of the samples had the same problem. Such sample showed one sharp, but thin band of 28S RNA, and the other was almost invisible. However, the OD value of this sample is the lowest one; thus low concentration of total RNA is a possible cause.

Amit Kumar Hi amit i am also facing the same problem

have you solved your problem ?