How did you measure these 8ng/µl concentration? Is this trustworthy? Or could it be that this come from noise, bad baseline adjustment, other nucleic acids, solvents, proteins...

How was the cRNA quantified? Have you also checked the electropherogram? Does it look normal? Have you tried a "negative-control" reaction (without adding RNA) to see what happens when the sample would be empty?

I measured RNA concentration by Nanodrop. Of course, other molecules can be presented in RNA samples. Low 260/280 and 260/230 ratios and also RNA concentration, which was measured by 2200 Tape Station and was much less than RNA quantity in Nanodrop, can indicate impure RNA. So, I think that RNA samples actually contain less pure RNA and whole transcriptome amplification could be helpful to increase RNA quantity. Can I use Ovation PicoSL WTA System V2 (Nugen) to perform it? Will cDNA amplified by Ovation PicoSL WTA System V2 be suitable for One-Color Microarray-Based Gene Expression Analysis? Or there are other WTA kits?

cRNA was quantified by Nanodrop as recommended by Agilent and checked electrophoretically (please, see the attached photo). RNA samples seem to be degraded. Or RNA have to look like this after labeling? Negative control reaction was not performed.

The PicoSL kit works well in our hands. I can recommend it.

Measuring RNA concentration of such tiny samples (as obtained from microdissection) is a difficult task, and OD measurements are not recommended. A fluorimetric mesurement would be better, and a quantification with qPCR would be best.

I can not say much to the attached electropherograms, ecpecially not without seeing a negative control (I assume that B1 serves as positive control).However, the profiles of B1 and C1 look considerably different, so I wouldn't conclude that the (specific) amplification of C1 worked. Just getting "something" that absorbs UV light or shows a band in a gel is a specific product ;)

One note to the PicoSL kit: the SPIA gives different results for the complete abscence of nucleic acids and for samples that do contain some residual molecules but not enough to be amplified. Here it is important to run "negative" controls with some molecules, but with a concentration below the lower limit of operation (see manual).

A final note: take care to avoid contaminations: SPIA produces DNA and is sensitve to carry-over contaminations. Better do amplification and all other steps in different labs.

Hello Evgeny,

I had a similar problem some months ago. I was isolating adipose-derived stem cells with a specific receptor. I was getting 100K cells using the regular RNeasy columns and my sample had a low 260/230 ratio. I measured the 230 absorbance of the RLT and RPE buffers and found that they are high, specially the RLT. Here are some extremely important points you should consider:

1) Reduce the amount of lysis buffer you use. The Qiagen protocol asked to use 350uL and I am using 100-250 uL. If you reduce the initial amount of salty buffer, you will get a higher 260/230 ratio. The RNeasy Plus Micro tells you to lyse with 75uL and then adjust the volume to 350uL with RLT. I advice you to avoid adding more salty buffer. Vortex vigorously your sample in RLT for homogenization. The volume of 70% ethanol you add should be the same as the RLT buffer.

2) I found out that my RPE buffer (kept at room temperature) was not working properly due to some evaporation. I figured out it is best to prepare it (ethanol + RPE) prior to use.

3) Make sure that your RNA samples are not degraded.

4) Try doing the low-input quick amplification with 50ng. This will increase your amplification and thus you will get a better labeling.

The 260/280 and 260/230 ratio should be as close as possible to 2. The salts interfere with amplification and with labeling.

Good luck!!

Dear Jochen, many thanks for your suggestions and comments regarding PicoSL kit!

If you have experience with the combination of PicoSL kit wit Low Input Quick Amp Labeling, could you please share with me the protocol? When should we use PicoSL? As I understand we have to use PicoSL kit instead of cDNA synthesis in Low Input Quick Amp Labeling and then perform cRNA synthesis, amplification, amplification etc. according Agilent protocol. Is it needed to amplify poly A RNA (from One-Color RNA Spike-In Kit) together with our RNA samples using PicoSL kit?

So, we will try to purchase PicoSL kit and to amplify our RNA samples.

Unfortunately, we do not have fluorimeter. As to RNA quantification by qPCR, I would also ask you to share with the protocol or with the link.

Thank you in advance!

We use the PicoSL for amounts from 50pg to 10ng totalRNA.

In the appendix of the kit manual there is a section about "Guidelines for Use in Microarray Experiments". We follow this. There is an application note for Agilent at http://www.nugeninc.com/tasks/sites/

nugen/assets/File/technical_documents/techdoc_agilent_appnote_01.pdf

Regarding the qPCR there is no special protocol. It is a standard qPCR. Use primers against a gene that is relatively highly and stably expressed (18SrRNA, bActin, something like that). If you do the RT with oligo-dT-primers, design the gene-specific primers close to the poly-A-tails. You may also consider specific priming in the RT. Then take an RNA sample of known (high, well-measurable) concentration and dilute it down to the range where you expect the microdissected samples to be. Make a dilution series around this range and perform a quantification with a calibration line, that's all.

I generally am not very concerned about a high precision (althouh I generally try to achieve the highest possible precision under feasable efforts, for sure). Plusminus a factor of two or three will not be a big problem for this purpose.

Thanks a lot, Jochen! Your recommendations are very useful for my study!

I will quantify RNA by qPCR and write the results here.

I can't open the link you sent (http://www.nugeninc.com/tasks/sites/

nugen/assets/File/technical_documents/techdoc_agilent_appnote_01.pdf). In Nugen website, I found Agilent Application Note #1 (http://www.nugen.com/nugen/?LinkServID=4A7438FB-E8AF-4BFF-A64A08D513C3C8D3), which you probably mentioned.

Dear Raquel, thank you for suggestions! But all attempts to increase 260/230 ratios of my RNA samples failed. So, the reason is an extremely low concentration of RNA from microdissected samples and the inapplicability of 260/280 and 260/230 ratios to such RNA as suggested by Jochen Wilhelm.

Evgeny, sorry for the wrong link. It was copied from the Nugen protocol I have and it seems outdated. Your link directs to a more recent document, and I think this is ok. Just take care that you would possibly need to adjust the abounts when you are using 8x60k arrays. We rescaled the amounts accoring to the Agilent protocols.

Dear Jochen, thank you! I will keep it in mind.

Dear Jochen, sorry for bothering you again! Could you please tell me what distributors do you use to purchase PicoSL kit? The matter is that in Russia, there are no distributors of Nugen products. However, we collaborate with the University of Heidelberg, and I think that I can purchase PicoSL kit with the help of my german colleagues. Thanks in advance for information!

I order the kits from the Europe office of Nugen. You find this and other customer support offices at

http://www.nugen.com/nugen/index.cfm/support/customer-support/

I'd hope that you should be able to order it directly. Otherwise, your German colleagues will surely get it under the above address.

Best of luck!

Thank you very much for all your help and useful information!