Did you treat your RNA with DNase?? If yes then maybe their was RNase contamination in your DNase and it chewed up your RNA if that is the case try using RNase Inhibitor when you treat your samples with DNase. Other than that I cannot think of anything else quiagen and promega are both reliable kits I have used them both without any problems.

Also the concentration of cells you are adding at the beginning of your extraction is crucial try using the recommended amount, if you add more then you sabotage your extraction or if you add less then you will not have a sufficient amount of cells to extract RNA.

And another tip try to double elute this increases your yield by 20-30%, add your elution buffer let it incubate for 5 mins centrifuge take the eluted sample and place it back to the column incubate for 2-5mins R.T and spin again. This always works for me.

I hope I answered your question.

@ Shailesh, actually extraction of RNA is very sensitive. Precautions must be taken against exogenous RNase include the use of plastic gloves, RNase free tube and pipette, autoclaving solutions after adding 0.1% (v/v) diethyl pyrocarbonate (DEPC; except Tris, which reacts), and heat the mortar pestle at 180°C at least 3 hours, chilled the mortar pestle with liquid nitrogen before grounding. You can used TRIzol™ Max™ Bacterial RNA Isolation Kit (ThermoFisher Scientific) for RNA Isolation. During centrifuge you should used 4 degree centigrade temp. Below link may be helpful for your research.

Dear Charalampos Filippou, Thank you for your tips I will surely follow these tips.

I have used oncolumn DNAse treatment and yes I need to try with Fresh aliquot of DNase.

I am using RNase Free water provided with kit for elution, do I need to use buffer for elution?

Santosh Kumar Bose Sir, Thank you very much for your suggestions and protocol.

Dear Shailesh Shiwajirao Sawant, RNase Free water is ok to use just make sure that is actually free of RNases, if it is part of your kit and is new then most likely is ok but if it is an old one then I recommend to use sterile milliQ water (be very careful though, use all precuations) if you're going to use it for qPCR later on.

I am using on column DNase too for DNA digestion and I prepare a mixture of 1ul of RNase inhibitor along with the DNase I and buffer to a total of 100ul for each sample before I load it on the column and I don't have to worry about RNases.

And one other thing I forgot to mentioned later, mix 2-mercaptoethanol in your lysis buffer prior extraction, this will inactivate and eliminate any RNases present in your sample, every 1mL of your lysis buffer add 20ul of 2-mercaptoethanol.

Believe me I know RNA extractions are very tedious ( I have been extracting Fungal RNA for too long now) and you must be very careful because RNases are everywhere even in the air and are very stable enzymes, but as long as you clean everything with DEPC water and RNase Zap (is your best friend when extracting RNA) and constantly changing gloves you will be ok.

I hope you all the best and good luck with your experiments.

Dear Charalampos Filippou Thank you very much for suggestions. I was thinking to use mercaptoethanol but I was not sure. Thank you I will check it soon

Dear Charalampos Filippou Thanks a lot. Results improved with addition of 2-mercaptoethanol.

Dear Shailesh. with the all recomendations to RNA manipulation take care, I recomend you store an aliquot of sample in ETOH 70% before to resuspend in water, The RNA is most stable in ETOH than in watern for more time in -80°C

Oscar Badillo Thank for your suggestion, after harvesting cells I store it in methanol at -80 .

Is it only now that you don't see bands? How about your ladder?

Willem-Jan Wollants Ladder is okay, I am using bleach gel for separation . Now RNA concentration is very low.