RT-PCR primer design and in silico PCR - (Jul/06/2008 )
I use Primer 3 to design cDNA primers according to the very useful guidelines as below, thanks for their contribution!
However, when I go for in silico PCR tool (UCSC), the results showed no potential amplicon of my target ? So, what can I do? My aim is just would like to check the present or absent of mRNA expression ..
If any of your primers is located on two exons (on the exon-exon junction), in silico PCR won't return any hit in most cases. If both primers are entirely within exons (two different exons, of course), you should get hits. You can use the PCR products returned by primer3 to do a blat search to see where your primers are located.
Thanks for your explanation . Hmm, I knew my primers location by using a sequencer software. However, I failed to amplify cDNA using primers desinged on the exon-exon junction last week. So, I thought can use in silico PCR to check whether can I amplify the cDNA target region, but it seems I was wrong.
May I know how can I confirm the primers whether can work or not? My target region (cDNA) consists of 4 exon, total size is around 500 bp. Can I employ multiplex PCR to amplify all four fragments in order to save template (cDNA commercial available)'s volume ?
If you are sure the mRNA sequence you used for primer disign in Primer3 is right, and you didn't get pcr product, the likely reason is that the gene expression is too low. You really need a positive control cDNA sample (for example from cell lines that express high of your gene) to tell you the primers are OK. You don't need to do multiplex PCR.