Dear Qiu Xuan Tan, 

If you do not want to use ethanol, you can try the kit RNA Clean & ConcentratorTM-5 from ZYMO Research (Catalog Nos.R1013 & R1014 (supplied with DNase I) R1015 & R1016), it is based on columns and it is very easy to use.

Hope it helps

I think you have two choices:

1) Let the water evaporate at 37ºC with the tubes opened.

2) Re-purify the extract again in the columns and elute with 25ul.

Is a 2-fold difference in concentration worth the risk?  The difference in Cq value sensitivity from a 2-fold concentration would be less than 1 cycle.  

Low yield from a normal cell input (as you have described) is usually indicative of having picked up an RNAse contamination during the extraction, unfortunately.  It might be worthwhile to check your RNA sample on a Bioanalyzer to ensure its quality before proceeding. 

qPCR is very sensitive to pick up very small quantities of RNA. The specificity varies depending on the detection chemistry or kit that you are intending to use. I would advice that you first quantify your sample and confirm if it is within the recommended range of the kit. Sorry I have never concentrated RNA i wouldn't help much with that. All the best

I you have access to a lyophilizer in your department....that should be perfect. 

If your RNA is intact (see suggestions above), you can try extraction by 1-butanol.

Reference: http://www.ncbi.nlm.nih.gov/pubmed/19146820

Simply add 1-butanol and mix, part of the water will go into a saturated mixture with the butanol, while still maintaining phase separation. You can then remove the butanol by pipetting, and extraction with diethylether.

precipitate in ethanol+ range free glycogen, then resuspend in require volume.

Before you do qPCR, you need do reverse transcription PCR, right? The demand of total RNA is 10 pg to 5 ug in "ThermoScript™ RT-PCR System" kit. 

Hi Qiu Zuan,

Column RNA purifications can sometimes result in loss of RNA sample - and that is perhaps what you have experienced. Hopefully your cells were not lysed before the pellet was placed in the RNAse-inactivating GIT-like-containing reagent in the kit. RNases could have degraded some of your RNA if the pellet was not processed correctly prior to application to the column.  What were your A260nm readings of the RNA sample(s)?  When in doubt, use the Trizol method for RNA isolations (2 to 10 million cells/1mL Trizol). Then, be sure to not over-dry the final RNA pellet.  But, if you do over-dry, add the desired amount of nuclease-free water (50 uL is fine), and then place at 60C for no more than 2 minutes to help resolubilize the RNA pellet, vortex briefly, spin down briefly, and proceed from there.  Be sure to load the same amount of RNA per each RT reaction as well (if making cDNA before qPCR - in a two-step approach).  I agree with many of the points in the above posts - and, if you can, do use a Bioanalyzer to see what your RIN values are for your isolates; this will help you in determining if you have degraded RNA or not - as spec or NanoDrop readings can give great A260nm and A260nm/A280nm ratios - but the RNA can still be of very poor quality. Aggressive lysis of your cell pellet in an RNAse-inactivating reagent preceding RNA isolation is key here. You need to stop RNases as early on in your protocol as possible (e.g., with Trizol, add the Trizol directly into the cell culture flask and scrape cells to collect, then vortex aggressively to homogenize). Not knowing more about your actual situation, this is as much as I can suggest.