Related to very first question, the answer is yes. You can amplify from one primer in intron and one in an exon for example. Considering that you DNase treated your RNA samples prior to reverse transcription, one can monitor the level of pre-mRNA this way (however usually pick up at very high cycle number). Product related to gDNA contamination should be exactly the same length (the gDNA contamination product is bigger when the primer where exonic and span an intron). Sometimes the exon/intron boundaries are not clearly define and/or it is possible than you have an alternaative 3' splice site. As Can Kucuk suggest, I would gel extract and submit this two band to Sanger sequencing to clarify the situation anyway.

hello

you can Allele ID for primer design. i think you cant amplify.

Thanks Sadegj, so my gel is amplifying the right produtc? Bellow it a gel with my products. Expected product is the band on the top.

Hi Aletheia,

Assuming that the primers are designed for q-RT-PCR and one of the primers is in an intron suggests that there may be some genomic DNA present in your cDNA sample and it may be giving the expected size amplicon. The best way to be sure would be to do gel extraction, and then TA clone and Sanger sequence the amplicon that gives the expected band.

Can

Hello Can,

Thanks for reply.

Regarding to your answer....If I had gDNA contamination like you said, the PCR product would be bigger. Around 360bp and it is not the case. But I got an unespecific band...

Related to very first question, the answer is yes. You can amplify from one primer in intron and one in an exon for example. Considering that you DNase treated your RNA samples prior to reverse transcription, one can monitor the level of pre-mRNA this way (however usually pick up at very high cycle number). Product related to gDNA contamination should be exactly the same length (the gDNA contamination product is bigger when the primer where exonic and span an intron). Sometimes the exon/intron boundaries are not clearly define and/or it is possible than you have an alternaative 3' splice site. As Can Kucuk suggest, I would gel extract and submit this two band to Sanger sequencing to clarify the situation anyway.

Hello JP ,

You are right, I DNAsed all samples before RT, and it is very reasonable the boundarie explanation, make sense to me. However, I have two questions:

1. My forward was all over the genome. I had unspecific alignments in many chr, except on the right place (see att.). Can he still amplify in this case?

2. If the primer can amplify even being in an intron, this could explain why I got inconsistent results? Using same samples and same control, my primer behaved different which used to drive me crazy....

Unfortunately, I can not sequence my products. I am done with my bench work. I am gonna present my results in 30 days :-)

Thanks a lot for your help. I really appreciate that.

1. You will get unspecific amplification only if both forward and reverse primer are aligning in close proximity. You can use UCSC Primer in silico to verify it by copy/paste your sequence http://genome.ucsc.edu/cgi-bin/hgPcr?db=dm3

2. I don't understand your 2nd question but if these primer pair are use for quantification purpose, I just re draw primer and I don't bother why it didn't work unless I am specifically interest by mapping the sequence region. What is the goal of the experiment?

Good luck with your presentation

JP, I insert my sequences on this link you suggested. No matches.

I also did a BLAT on the same browser. Only reverse matches...

This gene (PRM1) is the internal controls of my qPCR. All the targets worked fine, all of them amplified expected products, including the control even with extra band.

I really need to understand what's going on, otherwise none of my results are valid.

Thanks a lot for your reply.

In fact, whatever the BLAST give you, what matter is if you have a single expected product, particularly if you are relying on SyBr Green...

Does internal control mean reference gene (to normalize quantification)?

If you have other gene target that do not move a lot between control and experimental condition, then you could use this gene as reference rather than PRM1 that is giving multiple band.

Internal control is not a reference gene...Anyway, I have to do this.

Thanks for your help.

All the best, Aly

Hi Aletheia Souza,

My first question is - why do you have a primer on the intron? Usually for RT-PCR you want your primers on the exon - either flanking an intron or one that overlaps the exon-exon junction. I suggest that you consider re-designing your forward primer on the exon. Avoid the intron at any cost. It can amplify only from genomic DNA; not cDNA. When primers do not have the right target, they can produce spurious products. I would argue that the product you say is of the predicted size is not your true product because cDNA should not carry the intron - of course unless there is differential splicing.

Hi Sakthikumar,

My goal was design a primer for my gene (which is my internal control), and for this I used a sequence from NCBI. Both forward and reverse were supposed in a exon region. However, BLASTing the sequences on ENSEMBL, the browser showed that they are in a intron. It was not on purpose.

Thanks anyway for your reply, but I think that the point is not so simply.

Regards, Aly

my primers forward and reverse are on intronic region of my gene. i have ran a PCR and  found no bands on DNA Agarose gel .moreover if i designing my primer on my exon then half of y gene sequence is lost which is vital for my sequencing.please anyone help me