qPCR primers could work on the cDNA and not the gDNA if the qPCR primers span a genomic intron/exon junction.  The cDNA is made from reverse transcription of the mRNA transcriptome, which lacks intron sequences. Check to see where your qPCR primers are binding and prove to yourself that one or both ot the qPCR primers span a genomic intron/exon junction.

Hi Jack, thanks for your reply. However, the problem is the exact opposite. As I said, I managed to amplify the desired product (hole gene) from genomic DNA and not from cDNA. I checked the cDNA quality using known qPCR primers and I got amplification, which means my cDNA is ok. However, when I tried to use the cDNA to amplify the hole gene it doen't work. In other words, the same set of primers work for genomic DNA and do not work on cDNA.

Oi Julio!

That has happened to me before.Do you know the expression levels of the gene you are trying to amplify? It may have very low expression. You can try to synthesise cDNA from different plant organs or developmental stages. Or maybe increase the amount of cDNA template you are using.

Hope this helps you out.

Cheers! :)

Hi Julio,

Joana and Wilco already stated the first question you need to clarify: is there any expression of the gene in the tissue from which you isolated the RNA which was used to synthesize your cDNA template. If there is no information on this, it may be hard to get an idea about this (you would need to test different tissues, or treatments etc.). If there is no or very low expression it might be hard to amplify a product from cDNA. Mybe cDNA amplification might help in this case.

When you tested the cDNA quality with your qPCR primers, was the efficiency of the qPCR nice or was there any hint that reverse transcription was not very efficient?

Good luck with your experiment!

Gertrud

Hi everybody! Thanks a lot for all your suggestions! Concerning what was said about the gene being or not expressed in the conditions I am testing, that could be a good reason. However, I did a qPCR and noticed that the gene is expressed in the condition I am using to synthesize the cDNA. Wilco raised a good point when he asked about the transcription start deduction. Since I am working on soybean, I guess the deduction is correct. However, I cannot rule that out. In my last trial, I used concentrated cDNA and two sets of primers, one designed for qPCR and another for cloning. I loaded the products in a gel and the bands for the qPCR products were there in the right size, both for the cDNA (~140bp) and for the gDNA (~450bp). That is an indication that the cDNA is ok. When I used the primers for cloning, I only got a band for the gDNA. Any other suggestions?

Here is a pic of the last gel. In the upper lanes you can see the amplification for the qPCR primer (primer 15). In the lower lanes are the products of the reaction with the primers for cloning. From left to right is "cDNA RT (root treated)", cDNA RC (root control), gDNA, and negative control. In the right side is written the expected fragment size for cDNA or gDNA products. Notice that in my reactions aiming at cloning the hole gene, I have a strong primer band in the bottom in all samples. That could be primer excess or self amplification, I don´t know. I am working to solve that. 

You may have you CDS wrong. Blast your translated cds against the protein databases of other closely related species, that might give tou a hint. You can also try pairing your cds primers with your qpcr primers and try to understand if the begginong or the end of the sequence are well determined, in case you get a product of the right size. 

Thanks guys! I will re-start those experiments today and see what happens. 

Hello everybody, I think I found the reason why I cannot amplify the fragments. I have used a kit to synthesize cDNA that is not ideal for down-stream cloning. I used the High-capacity cDNA Reverse Transcription Kit from Applied Biosystems. I am still not sure but I think I don´t have the full lenght cDNAs I need. Cheers!