6 replies to this topic

[drwho]

Hi all

I hope you can help I have read many topics on this issue but thought I would ask for some help.

I am trying to isolate RNA from a small numbers of cells ~1000 (human samples).  In my trial experiments using cell lines I managed to get usable quantities and quality RNA using Guanidinium thiocyanate-phenol-chloroform extraction (TRIZOL) with an additional ETOH sodium acetate precipitation, also tried a butanol concentration set with good results.  I was able to repeat this in a number of differnt cells types.

However now I have tried it on the real samples I have good quantity and good 260/280 values however my 260//230 values are low ~1.3-1.5.  I am at a bit of a loss about what to do.  I will be unlikely to get more of these cells for a long time if at all.  

Any advice on how I can improve my 260/230 values.  I will want to use the RNA for standard real time PCR no arrays or anything like that.

Any suggestions more than welcome.  

Edited by drwho, 27 September 2012 - 11:25 AM.

[mdfenko]

you may not need to improve the ratio.

but, if you do then you can perform an additional chloroform extraction followed by precipitation with ethanol/sodium acetate.

[drwho]

Thanks for the reply.  If it were any other sample I would just give it ago but given the scarcity of my sample  I am canvassing for opinions!

Do you have experience of PCRs using RNA with low 260/230 levels?  I would be more conformable if all the values were the same between the patient samples ie equal error.  But they are different enough to make me concerned.

Cheers

[Trof]

260/230 level will be always low because the read comes primary from salts or phenol and there is some minimal amount of this always present, and since you have very little RNA, the 260 read is very low. It doesn't mean your sample is so "dirty" it just that the ratio of dirt and RNA in much lower. That may be the reason why the ratio is different between samples, do some of them have higher concentration?
You could get rid of the phenolic remnants by second extraction, yes, but question is if you want to go through that with your very limited amount of RNA, you will always loose some in further steps.

Are you doing qRT-PCR? You could measure expression of some stable housekeeping gene to see if it has any effect on measurement. But since you probably have very low concentration, you may not be able to put equal amounts to RT, so it may display bigger variation, that with normal concentration of RNA.

[drwho]

Thanks for the reply Trof

qPCR will be my final application.  I think I will proceed with the PCR rather than do any further clean up steps, as you are right I am likely to loose more sample.

The cells I am looking at a specific sub-population of cells, but I also have larger amounts of the whole population of cells which have been prepared in the same way but have good quality and quantity of RNA.  I was thinking of comparing the housekeeping genes between the populations, this should tell me if the contamination has affected my reactions and whether my data is any good.  I would be grateful for you thoughts on this approach?

Thanks in advance

[Occam's Maser]

If I might offer one further problem for discussion; I just yesterday discovered how terribly 260/280 absorbance quantitates DNA at low concentrations. I am extracting DNA (and hopefully RNA) from small numbers of FACS sorted human cells. Turns out NanoDrop/Vue grossly overestimate DNA concentrations. I was recommended the Qubit fluorometry system which worked well at my first attempt and gave results far more in keeping with the number of input cells (500) rather than 2-3 logs higher concentrations claimed by absorbance. I am now very suspicious of my "good" RNA readings from NanoDrop... sigh... Qubit has an RNA option too, which I hope to try. Good luck and please update us if your protocols all work out.

[Trof]

Logically there is still relatively the same caryover of certain things that affect 260/280 ratio, so if the DNA concentration is really low, the ratio will go bad. The same is true for 260/230 ratio even in much less limited samples, if you have calculated concentration aboud 300 ng/ul the ratio is much better than if the concentration is 10ng/ul.
Fluorimetry is more precise than absorbance of course, but has certain financial disadvantages. So if you need to measure low concentrated sample of have a very precise measurement, Qubit is definitelly a better option and there is nothing much shocking about that.

By the way, what was the Nanodrop value? Since the absorbance measurement in general is in my experience not reliable at all under 15-10 ng/ul, I wouldn't even consider using absorbance for measuring such low concentrated samples. On the other hand I once got a 2ng/ul concentration measurement for some DNA, and still managed to PCR something out of it, so just for telling me the sample is not completely negative it served it's purpose.