I agree with Enrico that 1.5 ng/nl is an unusual concentration unit. If you meant 1.5 ng/ul then that is low and likely is in the noise range of the nanodrop. If it is 1.5 ug/ul, then fantastic. It sounds like you might be worried about genomic DNA contamination. If you are, then you can DNase treat your sample and then reprecipitate the RNA. Alternatively you can make cDNA from the sample and then use transintronic primers over a short intron (about 50-100 bps) and run the product on a gel. If you have a longer band (i.e. 50-100 bps longer than what you would expect from the cDNA alone), then you have gDNA contamination. Hope that helps.

Dear Andrea, this is definitely too little RNA. You should get something in the range of 80-500 ng/ul. Something might have happened durgin RNA extraction. On the otehr hand, sometimes nanodrop is not very reliable, so I would run an aliquote in an agraose gel or measure it in another apparatus (e.g. Qubit, if you have one). If you wnat to try another extraction, go for the QIAGEN kit (for good purity) or TRizol (for a better yield).

Andrea:

Do you mean 1.5 ng/ul? If so, you got ~10 ng/ul mRNA (counting the dilution factor) and it seems reasonable (although on the low end) to me. mRNA is generally 1-10% of total RNA, so it seemed reasonable to proceed with rtPCR without dilution.

1.5 ng/nl in 1/5 dilution is a lot ( but I suppose you have done a 1000x mistake in units, see Enrico Tatti reply). Are you isolating PolyA+ RNA?

I agree with Enrico that 1.5 ng/nl is an unusual concentration unit. If you meant 1.5 ng/ul then that is low and likely is in the noise range of the nanodrop. If it is 1.5 ug/ul, then fantastic. It sounds like you might be worried about genomic DNA contamination. If you are, then you can DNase treat your sample and then reprecipitate the RNA. Alternatively you can make cDNA from the sample and then use transintronic primers over a short intron (about 50-100 bps) and run the product on a gel. If you have a longer band (i.e. 50-100 bps longer than what you would expect from the cDNA alone), then you have gDNA contamination. Hope that helps.

I Agree with Daria. Check really if you've got 1.5ng/nl that is equal to 1.5ug/ul and that is alot. Especially as you are talking of a 1/5 dilution as well.

Several years ago I also tried to isolate mRNA and resulted in very low or no mRNA at all. Since then, for cDNA synthesis I use total RNA. Gene-specific primers i design so they would be in different exons. However, I do DNase treatment after RNA isolation and clean up with RNeasy kit. For cDNA, we usually use starting from 500 ng of RNA.

Hi Andrea-I have couple of comments. If the 24G needle you use was not blunt, that could explain the foaming as well as a large syringe volume as it compares to a small lysis mixture volume. The amount of cell debris is little from 2 million cells. In regards to the amount of RNA, if indeed you were using the mRNA isolation kit then is a reasonable yield. If you were using a total RNA isolation kit then I would say your yield is very low. What I would do, if feasible, is to re-isolate from fresh cells with all these in mind. I wouldn't want to proceed further if not certain of the quality and the quantity of the RNA. If you have access to an Agilent Bioanalyzer you can easily evaluate the quantity and quality of your RNA using the RNA chip.

I don't think so it is best idea to dilute mRNA so much, it is better perform the cDNA synthesis and dilute if it necessary then. I agree with Inese if you use this RNA to real time PCR just use total RNA and design the primers on the exon/intron border.

I suggest to extract total RNA with Trizol first, then enrich the mRNA fraction using the kit. Consider that mRNA usually represents 3 to 5 % of total cell RNA. So even if it seems to you to have recovered small amount, be aware that you got rid of 95% of RNA that unlikely you are interested in.

Good luck

Hi, Andrea thanks 2 all in the thread I think u got the true answer for way out your experiment, I suggest you to isolate total RNA and from 1 ug of it use oligo dT primers for cDNA synthesis directly, remove DNA by Short DNAse treatment if required for you RNA isolation protocol. All the best.

The yield depends on the starting materials you have used for which kit ; as Invetrogen has 4 kits for direct mRNA extraction. However, the yield sounds reasonable to proceed for the RT-PCR step. If it failed you might consider re-extraction of total RNA followed by mRNA enrichment step. I prefer to avoid the shearing step and use DNase treatment on half of sample volume. It is also advisable to check the quality of RNA on denaturing agarose gel if you are interested in quantifying the expression.

Hi,

I agree with Lichinchi..

Andrea u did not mentioned the 260/280 ratio.. check it properly... concentration is not very important but quality also matters.. however the concentration is good enough... if kit is not giving proper result try TRIZOL method for RNA extraction.. apart from this also u can check quality of RNA by gel electrophoresis (In MOPS buffer)... and try to make cDNA at the same day to get better result..

Good Luck..

Thanks to all your answers! Only now have I checked them well.

First some comments: * I was, as most of you guessed, 1000x off - the concentration (of the 1:10 dilution) the nanodrop gave was of course 1.5 ng/ul. That's why I was worried, since it seems awfully close to the noise of the machine. * I didn't note the 260/280 ratio, though. * The kit is the mRNA MAG * We did use (a very old) DNaseI. * We didn't use the dilution for the cDNA library (only for measuring), but the original solution, and it was the same day, on the fresh mRNA.

I can add that I successfully used the same kit a year and a half ago, but didn't quantify the yield, so I'm not able to compare... Today we tried the same thing again. The cell pellet was quite big. When we pipetted the cells in the lysis buffer, we could clearly see the long DNA strands. This time we managed to not create any foam. After centrifugation, there was no cell debris visible, though. Should there be? The mRNA concentration was as low as last time according to the Nanodrop.

Anyway, this time we DID get a quite bright band on the gel after the PCR on the cDNA library from the positive control. The gene we're interested in seems more difficult to get, but it shouldn't be due to the mRNA extraction though. We'll try another cell line, and we'll see.

So again thanks for all the suggestions!