Hello, please could any one help me in choosing a gene to design a primer for it , there are many variants of the gene and I do not Know if it is right to use any of these variants instead of the complete sequence of the gene ? I want to do gene expression analysis only by RT-PCR.
Look to the attachment picture below :
Perhaps you can use a consensus sequence to design a primer that can capture most of the varients
That's right, I suggest you to select primers at the most 3' of the gene to avoid degraded RNA mis amplification, and using the qRT-PCR rules to design the primers (almost one primer across splice site...).
attached a pict of the gene and sequence to select the right sequence.
you can also use the designed primers from other studies at right of this link/picture (http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg19&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr4%3A110834040%2D110934118&hgsid=1256525837_D08TYExkNUZFIyJFiQZ0AZhCBw6S).
all the best
fred
Which variant are you trying to study? Is the expression of each type tissue-type dependent? or age-dependent? or some other factor?
You need to step back and think about what question you are trying to answer BEFORE you start designing primers.
Plus, you'll need a full set of controls. See what's in the literature, talk with your research mentor and PLAN OUT your experiment (number of samples, number of replicates, how you'll analyze the data, etc) BEFORE you mess around with primer design.
Good luck!
Heres the geneID link: https://www.ncbi.nlm.nih.gov/nuccore/444738180. Just pick the dominant isoform. These guys can help with a lot of primer design: https://www.yourbiohelper.com/
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