Hi, maybe you should sequence this product and blast it. Mabye it's contamination... Cheers, Nadine

Hello Nermeen,

Three possible reason which strikes to my mind for your problem are:

1. Genomic DNA contamination in your RNA sample (Have you kept any control for RT-PCR which can detect splice variant? If that is also behaving in same way?) You can keep one PCR using the problematic RNA as template. If the preparation is clean it wont give you any amplicon whereas you would get a 500 bp amplicon in case your RNA is contaminated by genomic DNA.

2. If your RNA preparation is clean then there is a possibility that your native gene derived trancripts are not splicing properly. If that is the case, I feel, it would be an interesting to study why and how transgenesis might lead to differential RNA processing.

3. There might be a third possibility too. Many of the transcripts spliced differentially in different tissues. In case your transcript is of that kind then your observation might be quite natural. For example, say your native gene encodes a transcript in Tissue 'A' and also in Tissue 'B'. But further, in tissue 'A' it gets spliced whereas in tissue 'B' it does not. Now since you have a transgenic containing the spliced variant (cDNA that you have transformed), you would naturally get only 200 bp amplicon in tissue 'A' whereas both 200 bp and 500 bp amplicons in tissue 'B'.    

Hope this will help you. Best wishes

I used also water control, and DNA from negative sample.

water control did not show any fragment but DNA from negative sample showed the fragment (500 bp) as non-transgenic c DNA.

Can you elaborate a bit more? Do you mean non-transgenic by negative sample? If it is so, then it is quite expected to get 500 bp amplicon. I would still suggest you to set a PCR with RNA sample to be sure about any possibility of genomic DNA contamination. if you don't have problem of funding, then you can try Nadine's suggestion too. It will give you very specific answer regarding the source of that 500 bp amplicon. 

Okay, thanks. I will.

I did mean that the  DNA from negative sample (non transgenic) as cDNA from negative sample (non transgenic) showed 500 bp but transgenic some tissue showed only 500 bp  and other tissue showed only 200 bp and other tissue have both :). 

Hi Nermeed, It sounds like you may have genomic DNA contamination in your samples.

Can you clarify if you did the following?:

- extract RNA from all of your samples first?

- then did you make your cDNA library using some sort of reverse transcription?

- and then you set up your qPCR with your samples and a no-RT control (which is just RNA as a template)? 

Did you design your qPCR primers to run through an intron-exon boundary in your gene?

These would really help in troubleshooting your problem.

I agree with the earlier answers. It is essential for RT-PCR assays to include the no-RT controls for each sample. In case these show the 'genomic' band, you have genomic DNA contamination. When not, there is a difference in splicing between samples/tissues. I had this problem once using overexpression of the genomic fragment (70bp intron), seeing both spliced and unspliced product, but only in lines with strongest expression. Splicing may vary between tissues and be part of the regulation of the respective gene.

Hi Nermeen,

Am I correct for your question:

1. Your organism has this endogenous gene (500 bp), you produced cDNA (200 bp) from this same gene. You then transformed the cDNA into the organism you are studying for overexpression. So now the transgenic organism should have both the endogenous gene (500 bp) and the cDNA (200 bp).

2. So, (1) when PCR the non-transgenic organism, you should get a band (500 bp), and (2) when you PCR the transgenic organism, ideally, you should get two bands, the endogenous gene band (500 bp) + the cDNA one (200 bp), if you used a pair primers designed to amplify both of them at the same time.

3. Your observation of the PCR results from your putative 'transgenics':

(1) With both 200 bp + 500 bp: this is expected (way to go)

(2) In some tissue, you observed only 200-bp band was amplified. One of the possible reasons is that when a common pair of primers were used to amplify both 500-bp and 200-bp fragments, the 200-bp fragment will be preferentially amplified, so you only saw the 200-bp band.

(3) In some tissue, you only observed the 500-bp band. One of the possible reasons is that either the line is an escapee (non-transformants) or it is chimeric with both transgenic and non-transgenic tissues and you obtained the non-transgenic portion of tissue for PCR

thanks so much Dr. Yuan-Yeu Yau

I often found that with genomic DNA. could you have any paper or site can I use it to confirm that  because most of the transgenic showed only one band as positive and few of others showed two band.

Hi Nermeen,

To draw a concrete conclusion that "most of your transgenic showed only one band as positive and few of others showed two band", your need to be pretty sure that your PCR conditions can be used to amplify both fragments properly.

Nermeen, are you using ONE common pair of primers to try to amplify 2 fragments (one long: 500 bp and one short: 200 bp) in one PCR reaction?