Hi, I never used the RNA/Protein kit, but I used the Nucleospin RNA II before and it worked really great.

First question, what is the concentration of the RNA you get at the end, and what is the quality (OD 260/280 ratio) ? You said you paid attention to the right conditions and temperatures, so I'm assuming it should be ok.

If you get a lot of RNA, and with a good quality, then maybe the problem is just the visualization on the gel. Are you sure you use RNAse free reagent to run the gel ? RNAse free water to make the buffer, RNAse free tank etc ? If you run your gel for 50 min and it contains RNAse, no wonder that you see only one band. An alternative is to run the gel for a very short time (15-20min) , and see if you get a nice separation of the ribosomal RNA bands.

Good luck,

Best

HI Mohammed

The nature of Ladder used by you is not clear in the photograph.....In my opinion there is something wrong in your protocol and your isolated RNA seems to be degraded very fast......if it is true you very often see degraded RNA running like a low molecular band..kindly compare the ladder band close to the 'suspected RNA single band'...and conclude if it is degraded RNA

bye

Thanks to you all. I think it may have to do with the work area for casting the gel and probably visualizing the band, based on your suggestions. The work area where I did the isolation was really clean; I change gloves average of 5 times and always used 70% ethanol intermittently. However, I cannot say the same for the gel casting area. I will repeat and see as it goes, taking all suggestions into consideration. Thanks again

I would suggest try to clear your area including electrophoresis unit with RNAse Zap and also you might want to spray little bit on your gloves before using any of this reagent.setup. I would also recommend DEPC treatment for electrophoresis unit as well as all plastic wares. You can also use DEPC water for preparation of buffer and other reagents.

I hope this helps.

Seems you have used less amount of EtBr while making gel. Quantity of RNA is less and also it is degraded. Use fresh opened packets of eppendorf tubes and tips and try to isolate RNA and running gel as fast as possible for better result. Small degradation is acceptable if upper 2 bands are visible while they are not in your gel picture. Avoid using DEPC treated water which was previously used several times.

Hi,

I have a doubt, what kind of tissue did you extracted?

I had the same problem extracting RNA gonad tissue from teleost using TRIzol method. But suprisingly the extraction was correct!. I compared different tissues extracted in the same batch and the RNA quality of the other tissues (liver) was correct except for all gonad samples. I realised that the band was 5S rRNA! The amount of the 5S was so high, and 18S and 28S RNA peaks were completely dissapeared. It can occur as well in your tissue.

Please read the following article where it is explained more in detail:

Diaz de Cerio O, Rojo-Bartolomé I, Bizarro C, Ortiz-Zarragoitia M, Cancio I. 5S rRNA and accompanying proteins in gonads: powerful markers to identify sex and reproductive endocrine disruption in fish.Environ Sci Technol. 2012 Jul 17;46(14):7763-71.

Hope it helps,

Oihane

Thanks Oihane and Gael.

I seem to agree with Gael. The last time I used TRIzol to extract RNA from 8 tissues of a freshwater species, I had no problems at all and the extraction was done at room temperature. In fact, over 10 freeze-thaw circles later, the integrity of my RNA was still intact. I opted to use the kit because it would make things quite easy based on the number of samples I now have but that is not to be, because I seem to have lost the entire RNA. The kit never said anything about temperature but I will try it though.

Oihane, I downloaded the article you suggested and will read through and see how it helps me. However, if the single band I am noticing is that for 5S, then how could I view the 18S and 28S?

Thanks.

Hi Mohammed,

I have also used Nucleospin Kit to extract RNA from fish and molusc liver and digestive glands at room temperature withouth loose RNA integrity.

Answering your question about 18 and 28S rRNA; Using Agilent bioanalyser in some cases I was unable to see the other 2 bands, it only detected a peak around 120 bp. So, I am so sorry , but I cannot help you visualizing the other 2 bands, 5SrRNA was at least the 75% of the total RNA.

One more comment, appart from gonads a colleague also detected the single band in rat brain tissue.

Regards,

It might be a case for stored samples even if lysate/sample is stored in -80 C, I used to face same problem. Try using fresh pellet and lyse under harsh conditions.

I am also getting the single band, may be because my sample (rat liver tissue crushed in liquid N2) was stored in -20 C for 2 years. I have just tried to isolate the RNA. But still, I am confuse whether it is the band of RNA or something else. 

Please suggest me, what is actually present (bright band like structure)

above the well.