Hello,

I would like to check my gene structure when design rt-pcr primers to contain some intron region in the amplified region. Please let me know where is the best place to get such information.

thank you.

ZW

Hi ZW,

This is what I usually do (maybe there are better ways)

I. first get a cDNA sequence from NCBI. I only use Refseq if available. RefSeq represents the most reliable and unique cDNA sequence for a gene. Because searching nucleotide database is not effective (you may get hundreds of sequences for a gene) so I will go to LocusLink at http://www.ncbi.nlm.nih.gov/LocusLink to find the gene locus of my interest, then scroll down to the RefSeq section to the mRNA sequence whose accession number usually looks like NM_xxxxxxx
2. Copy the sequence to a program to design primers. Follow primer design rules and design the primers. Choose the best ones.
3. Copy the amplified region of your desired primer pair and then go to UCSC genome browser at http://genome.ucsc.edu/cgi-bin/hgBlat and paste the sequence there and do a blat.
4. Check if your product spans any intron. In the blat result, your sequence (mRNA) and genomic sequence will be colored differently.
5. Or you can first do a blat to know where exon-exon junctions are and then do primer design specifying a target region corresponding to the junctions
6. Another purpose of Blatting your sequence against genome database is to check if your desired amplified fragment has any homology for other regions besides the gene of your interest. The Blat results will tell you that.
7. Lastly, you can go to NCBI Aceview at http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html or Ensembl at http://www.ensembl.org, both sites privide detailed information on mRNA splicing.

Edit: Sept. 6.
UCSC also provides an in silico PCR tool which allows you to input a pair of primers and retuns potential amplicon targets in the genome. It is useful for checking off-target amplification of your Rt-PCR primers. You can find the tool here: http://genome.ucsc.edu/cgi-bin/hgPcr

Hope it helps

Agree.

I have a question:

Which part of a cDNA sequence (5' UTR, 3' UTR, ORF, or any motif region) will you choose as amplifying target for RT-PCR, if they satisfy all the requirements (uniqe in genome, average GC, etc)? My feeling is they should all be OK for expression study. Am I right?

Kawaka

Is it right???

Hi All !

Notice that to study gene expression, the best is to design primers on two different exons, thus you obtain something specific for mRNA/cDNA, because you can distinguish cDNA from genomic DNA

Take a look here... this could help !
http://www.biocompare.com/techart.asp?id=799

Hi ZW,

first a warning: I'm from the company making this software.

But you could try www.probelibrary.com/adc which designs intron-spanning real time PCR assays on the fly. Only, you have to use Probelibrary Probes for these assays.

I would be interested in knowing your opinion of the tool...)

Søren

Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark

www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes

Kawaka wrote:

I've had some problems with RT-PCR when using primers in the 5'UTR region. A gene may have different transcription initiation sites in different tissues, maybe even in a same cell line under different conditions. As an example, I was not able to amplify a gene from total pancreas RNA using the upper primer in the 5'UTR (nucleotides 69-80 of the RefSeq sequence) but the same primers worked wonderfully on liver RNA. I changed the primers to the exon2-exon3 region and then I got similar levels of amplification from liver and pancreas. It is not a case of alternate promoters, the exon 1 is the same in both tissues, it's only that different transcription start sites a few nucleotides upstream or downstream may be used preferentially in the two tissues.
Hope this helps. Cheers.