This is a bit unusual, few question I have to know before any suggestions -

A.How did you extract your RNA ,  by manual trizol or any kit base method, have you checked 260/230.

B. How old were your RNA and cDNA synthesis kit.

C. Will you try Invitrogen superscript-III cDNA synthesis kit yet.

Rest all looks good if possible check RIN values for your samples by Agilent Bioanalyzer.

With best regards.

Thank you very much for your answer.

A. I have done RNA extraction by "Ribospin Plant (plant total RNA purifiction kit)" - GeneAll but I tried also "PureLink RNA mini kit" - Ambion, the quantity of RNA is similar. I haven't checked 260/230.

B. RNA and cDNA synthesis kit are new, they will expire on 2017.

C. Thank you for your suggestion, I could try it.

Best regards.

Hello Francesco,

Do u use random hexamer or poly T tail oligonuleotide in your kits. If u use poly T tail oligonucleotide it will convert only your mRNA. mRNA accounts only 7% of your total RNA. If your targeting mRNA, get a kit which purify mRNA from total RNA and then you can convert that to cDNA. If you want all RNA to be converted then use both random hexamer and  poly T tail oligonuleotide.

Hope this helps, good luck.

Hi and thank you for your answer.

I usually use both random hexamer and poli T tail oligonucleotide together!

Hi Francesco

You most likely have a sample contaminated with an inhibitor. Since you used Ribospin Plant I assume you are working on plant RNA. If that is the case you might have a carbohydrate contamination (but since you haven't checked 260/230 or 260/280 it may also be phenolic molecules, you should check both ratios).

You should try an inhibition curve by titrating your RNA and check if you get in the end a linear Ct vs [RNA] ratio. If not you should repurify your RNA or choose a different method. If you are indeed working on plant RNA take a look at the article below.

http://www.ncbi.nlm.nih.gov/pubmed/22736512

Hi Andre,

thank you for your answer. I will read the paper that you have suggested to me.

Yes, I am working with plant RNA.

I will check 260/230 and 260/280.

Anyway I have checked through Qubit the total absence of protein and a very low level of DNA contamination

It is counterproductive to measure synthesized cDNA. The amount is too small.

Are you sure it is not the RT-PCR problem? Do these primers work well on other targets? Is it possible that this gene is not expressed highly in these plants?

I would try another housekeeping just in case.

Hi Oleksandr,

I have read some articles where they use 100 ng of cDNA for the qPCR and I haven't this quantity.

I am sure that my primers can work, anyway I will try different housekeeping and I I will check 260/230 and 260/280.

Thank you very much.

Hi Francesco,

This is typical of carbohydrate contamination.

You should always check ratios otherwise going further is useless.

And you will never know if your qPCR is reliable until your perform an efficiency test.

My method for mammalian mRNA is a double extraction with Trizol LS.

Good luck !

Thank you David.

I'm going to check  260/230 and 260/280. Which is a correct value for the RNA without contamination?

2 for both !

Depends on the following experiment, approximations are tolerated.

For instance, if you perform classical RT-qPCR, a 260/230=1.8 is good enough.

However, for microarray pre-processing, you have to be strict, but a bioanalyser is classically used for this.

Cheers

Hello to everybody.

I have read that the ratio of A260/A280 of my Ribospin Plant kit should be 1.8-2.2.

All of my samples are in this range, so I think that there isn't a great contamination. Furthermore I have checked also A260/A230, you can find the values in the tab. What do you think? My problem is the contamination?

Cheers!!