Hello,

First of all, by looking at your gel it seems that your RNA is free of any DNA contamination. However, you can include DNase treatment while extracting RNA. How much RNA did you load? Usually, 0.5 to 1ug is recommended to get a decent band. Also, how much ethidium bromide did you add? I recommend you use at least 10uL of 10mg/mL ethidium bromide for 100mL gel.

Hope this helps.

Best!

Hi alaa

your gel seems not so bad, I've seen worst.

percent and gel staining are import points, but sure you get some degradation of your RNA. You therefore need to follow right one purification kit recommandations, in addition to temperature and time respects.

stay safe

fred

Thank you for responding to my question.

Yes i did the DNA digestion step.

The RNA samples i have loaded were diluted samples (50ng/Microliter) i used 4 Microliter sample + 4 from loading buffer, (Norgen 2x loading buffer).

Another issue that i loaded RNA ladder in the first lane but I can't see it (1kb RNA Ladder - Cat. 15003).

I think your samples are free of DNA contamination. However, the intensity of 28S rRNA band is usually 1.5 to 2 times that of 18S rRNA band in best extracted RNA samples. Looking at yours, both the bands seem to have equal intensity with 28S band having lower intensity than usual. Also towards the lowermost part of gel you could see faint smear. All this is telling of RNA degradation your sample could have suffered during isolation process. Also, you could try TRIZOL extraction which always has yielded results better than many kits in my experience. Instead of formaldehyde gels, RNA samples can also be resolved and visualized on 1.5% agarose gel in 1X TAE with 0.5ug/ml EtBr.

All the best :)

Thank you so much for the detailed explanation.

* is it possible to carry microarray experiment with such quality of RNA.

Note: I did the extraction using trizol and chloroform, following i used the RNeasy kit from qiagen.

Again thank you so much for your comment.

After repition.

largelly better, but you still have degradation, mostly associated on the type of samples, not the purification solution you used. as an example, samples from FFPE tissues will always give you the badest gels.

all the best

fred

Thank you Mr. Frederic for your comment, RNA were extracted from fresh tissue samples.

Almost certainly those bands are ribosomal- remember that 90% plus of RNA in the cell is rRNA. With respect to microarray analysis- remember that most of the hybridizable material in some of your samples is rRNA- and across the gel the recovery of rRNA to other RNA is inconsistent. You may get a qualitative hint- but quantitative analysis will be problematic.