Designing primers - What is more important Ta or primer's Tm - (Jun/13/2011 )
I'm going to perform qPCR with SYBR Green for gene expression analysis. I intend to run my reactions using universal cycling (5 min 95°C + 40x<15s 95°C + 1min 60°C>) and I'm trying to design my primers the best possible way.
About what should I be more corcerned, the Tm of the primers or the theorical Ta for my assay? Tm and Ta should be arround which values??
If anyone could give some advanced primer designing information, I would be thankful!
you can design your primers either through NCBI primer blast or Primer3 output using the default primer settings..,see to that you dont exceed beyond 200bp product size and either your frwd or reverse falls along the exon-exon junction..
Tm depends on the method of calculation. Using primer3 default settings I design primers with Tm 4-5 degrees higher than Ta. So in this case set the optimum for 65 degs for Ta of 60.
PrimerBLAST uses primer3 algorithm to find primers and calculate parameters, but it uses different default Tm calculation, you have to change in Advanced parameters the Table of thermodynamic parameters from SantaLucia to Breslauer to get same values.
It doesn't mean the other one is wrong or something, only that my 4-5 deg rule doesn't work with the other, because it shows considerably lower Tms for all primers.
I can suggest you to use this tool in order to obtain qPCR primers with good quality and specificity,: http://updepla1srv1.epfl.ch/getprime/. This user-friendly plateform uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally demonstrating their high quality and demonstrating high transcript specificity in complex samples. Until now, you could retrieve primers in a high-throughput fashion for all Homo sapiens, Mus musculus, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio genes in assembled chromosomes annotated in the Ensembl database.
All the best with qpcr!