Hi Ragaa,

Here are the following points

1) Do RNA isolation very carefully. Use sterile - RNA free conditions. Use RNAse Zap.

2) Water: Use RNAase free water. Buy it from company.

3) Use new gloves each time and do RNA isolation in Chemical hood.

4) Check 2ug RNA on agarose gel. You should see three bands. Check this image

https://www.google.com/search?q=RNA+on+agarose+gel&client=firefox-b-1-ab&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjRlbbO_ObdAhVyNX0KHT_ED4IQ_AUIDigB&biw=1440&bih=701#imgrc=lG9HS_YHtRyRPM:

Smear represents RNA degradation and cDNA multiple species.

Good luck !

Dear Amanda

RNA appears as obvious bands on gel agarose but cDNA always appears as smear on gel.

Ragaa, in fact the smeary appearance of cDNA on gel is not a problem, it is normal behavior of cDNA, so there is no need to overcome this issue.

This smeary appearance is usually because of the use of random hexamers and / or oligo DT primes in your cDNA kit. Whatever cDNA is generated from such primers, it will be various sizes, especially by random hexamers, which bind to various spots at your RNA and generate various-length products, which are not heavy enough to give you distinct bands, unlike in genomic DNA case.

So, it is absolutely normal. No worries at all.

But, if you use GSP (gene specific primers) on your RNA to amplify cDNA for a certain gene, then you will see distinct sharp bands on gel and now there will be no typical smeary appearance, because of the enormous uniform amplification of that target gene only from your entire RNA content.

So never check the quality of cDNA on gel and never check cDNA quantity on Nanodrop, these both are wrong approaches, and also you can save lots of your time and resources by not doing this. Simply check your cDNA quality by running any good housekeeping gene through normal PCR or by qPCR.

I hope it is clear now....

Regards...

Thank you so much for your clarification Jawwad.

Jawwad Ahmad , your answer fantastic, i am suffering from the same problem , but now i am happy after reading your answer. regard .

شكرا لك دكتور جواد.

Dear Jawaad, I think it is a very effective suggestion for me as well. I am so happy to get such descriptive answer. Thank you so much.