1. For concentration determination, Nanodrop becomes unreliable around the 30 ng/uL mark. Try a fluorescence based sensitive method like Qubit?

2. You don't specify the volume you have, but if you want to verify integrity rather then purity100 ng is plenty of RNA to run even on a standard agarose gel stained with SYBR green dye! If you use narrow wells you can cut it further down, and using SYBR Gold your sensitivity will go up by 5-10 fold - I visualised 1 ng of single stranded RNA in a denaturing gel before.

3. Real-time PCRshould be internally normalised to a reference gene anyway, and can successfully be used even in part-degraded material (we do it for biopsies, for instance).

Good luck!

In case it is a precious sample, I would do the cDNA with the maximum volume of RNA available for the reaction and then do a functional test - run a GAPDH (or other housekeeping gene) only. If you are within 2-5 cycles (Ct) from what you get normally, you will have a sucessful PCR. In case it is not a precious sample (you can get another one easily), I would re-extract the RNA with more cells/tissue.

In my lab, we routinely section brain slices and punch 1mm tissue cores from discrete neuronal populations. Downstream applications include qPCR, microarray and RNAseq. From a single brain, we extract ~20ng/ul using Trizol and Qiagen cleanup kits, and use half of this as the basis for cDNA synthesis ahead of downstream molecular approaches. You won't be able to resolve this on a gel, you will need to use the nanodrop within its functional range (0.1 - 1) and check with the Bioanalyzer (in your optimisation of the extraction). I take it your 260:280 is good??? 

The low 260:230 implies a carryover of salt/phenol/carbohydrates in your prep (see link below) and these absorb at 230nm (affecting your ratio). Make sure that you are taking only the aqueous phase (and not the interphase) into your clean up and that you are following the protocols to the letter.

http://www.qiagen.com/gb/resources/faq?id=c59936fb-4f1e-4191-9c16-ff083cb24574&lang=en

1. For concentration determination, Nanodrop becomes unreliable around the 30 ng/uL mark. Try a fluorescence based sensitive method like Qubit?

2. You don't specify the volume you have, but if you want to verify integrity rather then purity100 ng is plenty of RNA to run even on a standard agarose gel stained with SYBR green dye! If you use narrow wells you can cut it further down, and using SYBR Gold your sensitivity will go up by 5-10 fold - I visualised 1 ng of single stranded RNA in a denaturing gel before.

3. Real-time PCRshould be internally normalised to a reference gene anyway, and can successfully be used even in part-degraded material (we do it for biopsies, for instance).

Good luck!

Thanks everybody for your help and suggestion.

I agree with all of the above

Yes in my experience the Nanodrop becomes unreliable below about 30ng/ul and Fluorescent dyes are much more accurate at these low values: This is why at such values the peak on the nanodrop @ 260nm is quite low compared to the baseline, implying low signal to noise. I have also personally noticed that 260/230 ratios of less than 1 potentially implying salt and in particular GTC contamination from RNA purification (which if true would inhibit RT) are actually misleading

The good news however is that even @ 30ng/ul the actual concentrations are consistent enough to be able to perform sybr green real time PCR: Any real variation in sample concentration leading to slight discrepancies in amplification signal will also be true of your housekeeper like GapDH and thus corrected for by normalisation to the housekeeper as part of expression analysis

Thus, failing the good advice above - or if you cannot assay samples by fluorimetry -  I would try Sybr green qPCR with cDNA at these concentrations. I routinely do but the first thing I would try is gel based RT PCR to validate your cDNAs but you will also be able to see if the housekeeping amplicon intensity varies much from sample to sample; In my experience it actually has not

In my experience you can use as little as 100-200ng of Total RNA based on the above nanodrop reading, i.e. add 3-5ul of RNA to your cDNA reaction, perform RT and then check by PCR with hosuekeeping gene such as GapDH

If you are really concerned about RNA concentration and valid readings when low than simply precipitate RNA: You will concentrate and that will render nanodrop readings more accurate. To ppt

Add 1/10 x vol of 3Msodium acetate pH4-5

3 x vol of Ethanol

1ul of 20mg/ml Glycogen

Incubate for 1 hour @ -80C

Then 1 hour -20C

Spin 13,000rpm 20min

Remove most of suernatant

Dislodge the pellet with 70% ethanol and vortex (to desalt)

Spin 13,000rpm 5 min

Remove all supernatant; air dry for ~15min. Resuspend in 1/3 of original volume such that samples @ 30-40ng/ul become > 100ng/ul. Nanodrop readings are then accurate, i.e. Steep peak upto 260nM and a 260/230 ratio < 1.0 implying low salt contamination

Good luck !!