You will have trouble doing RNA input of 1 ug for max 20 uL reaction as 75% (15 uL) of your reaction mixture is RNA, giving you insufficient volume for RT enzyme, buffers and other components. 

Perhaps you should lower your RNA input (

Aimilia Lydia Kalafateli, You may try to reduce half of elution buffer you added. Alternatively, you may reconstitute it by running into gel, then cut it out, dissolve it back and extract them back with lower amt of elution buffer by using a kit. I forgot the name of the kit. pls check. However, if you still have much of sample, better use your existing kit and try to use lower amt of elution buffer to 15 ul instead of 30 ul you added.  

Thank you all! I will look into your suggestions!

Best,

Lydia

I am agree with previous comentaries, you can do cDNA synthesis (depending on the RT enzyme) with 10 ng of RNA. I recomendo you to titrate different volumes of your template RNA, if I were in your place I would try from 10 to 500 ng.

On the other hand if you want to concentrate your RNA, one thing you cand do is to precipitate it with isopropanol at -70ºC overnight, then respuspend pellet on 10uL RNAse free water.

Dear Lydia,

I would also try to reverse transcribe with a lower RNA amount. I think it is the safest way, since the more you handle with RNA the more possible it is that it gets degraded. If you want to stick to your RT protocol though, concentrating the RNA would also work.

I can recommend the RNA Clean & Concentrator™-5 (Zymo Research). It works well and you can elute the RNA in as little as 6µl. I have always been critical about evaporation of RNA samples because of the possibility of RNAse contamination, although I know that some people do this successfully. 

Good luck!!

Dear Aimilia Lydia Kalafateli,

You should concentrate your RNA by lypholization as mentioned in above suggestion, if it comes under required volume of RNase free water, then proceed with your kit. Otherwise, other option mention above by Francisco.  

Dear Aimilia, for such case you can use the full capacity of your cDNA kit, like if it is suggesting that use maximum 10 uL RNA volume in a reaction (regardless of its concentration) so add 10 uL RNA sample for the RT step and don't worry about any recommended RNA input value (500 ng or 1 ug etc). RT kits always describe in their protocols about the maximum allowable RNA volume per reaction. Run the cDNA RT step with RNAse inhibitors (usually 1 uL per reaction) and purify that cDNA further by cDNA purification kit (both are easily available) and run your cDNA undiluted and diluted (1:10, 1:25, 1:50) both, on couple of HKG best primers you have in stock. HKG amplification above 25 ct declares poor quality of cDNA, which reflects also bad or very low RNA at startup. It is a well-tested method with acceptable yield in terms of cDNA.

If you can afford, try to get SS IV enzyme cDNA kits, otherwise, the above method will get you good results. And never ever quantify cDNA by nanodrop or spectrophotometrically, it is useless and baseless and can make you hypertensive, which of course you don't want.

You can further have a look into my previous answer;

https://www.researchgate.net/post/How_low_of_a_concentration_of_RNA_can_I_use_for_cDNA_conversion

Regards...

one way to go would be to precipitate the RNA with ethanol (5 ul Na Acetate pH 5 plus 125 ul ethnaol) pellet the RNA and resuspend it in a smaller volume.