Hi,
I am trying to design a custom TaqMan® Gene Expression Assay for the first time and am having issues with some of the genes that I wish to include. In some cases, it tells me that the best coverage assays are in the 5' UTR of certain transcripts and in other cases it tells me that both the primer and probe map within a single exon. Has anybody used these before and had any issues?
Many thanks
As long as you know what region you are amplifying; that is a good start. And, remember, you can force primer-probe designs into regions that should not [thermodynamically] work, but for inexplicable reasons, they still do. So, do not trust all of the existing Tm algorithms all of the time when doom and gloom seem to loom. Many times, the Tm algorithms employed are dead wrong and your assay will work just fine if you merely forge ahead. Complete knowledge of your sequences of interest is a must - as is any/all BLAST-proven fidelity of your primer and hydrolysis (TaqMan) probe sequences for your target of interest. Amplifying a particular exon has its advantages and disadvantages - but, is still informative nonetheless. If the exon you are amplifying is common to all splice variants, that could be a useful approach. Conversely, perhaps the very exon you are specifically targeting is the most important story anyway - for that transcript family. E.g., for VEGF, the 9 sister variants have very different personalities, and it might very well be (per tissue type) that the 'average' effect of all 9 personalities at any given moment decides what "VEGF" is doing in that tissue (a spectrum ranging from angiogenesis to anti-angiogenesis; 0 to 1; 'on' to shades of 'off'). It is a very tangled web - but all pieces are crucially informative to the entire active picture, per tissue (even cell), per instance. E.g., If the anti-angiogenic isoform of VEGF seems to predominate at the mRNA level, then one might assume vessel formation is being disallowed at the protein level. So, as might be expected, ultimately, only tangible protein analysis will help spell the truth nonetheless; since expressed/translated functional protein (as far as modern dogma still largely implies) is the final, intended poetry of the DNA code anyway. Gamble the pieces carefully, and interpret away.
As long as you know what region you are amplifying; that is a good start. And, remember, you can force primer-probe designs into regions that should not [thermodynamically] work, but for inexplicable reasons, they still do. So, do not trust all of the existing Tm algorithms all of the time when doom and gloom seem to loom. Many times, the Tm algorithms employed are dead wrong and your assay will work just fine if you merely forge ahead. Complete knowledge of your sequences of interest is a must - as is any/all BLAST-proven fidelity of your primer and hydrolysis (TaqMan) probe sequences for your target of interest. Amplifying a particular exon has its advantages and disadvantages - but, is still informative nonetheless. If the exon you are amplifying is common to all splice variants, that could be a useful approach. Conversely, perhaps the very exon you are specifically targeting is the most important story anyway - for that transcript family. E.g., for VEGF, the 9 sister variants have very different personalities, and it might very well be (per tissue type) that the 'average' effect of all 9 personalities at any given moment decides what "VEGF" is doing in that tissue (a spectrum ranging from angiogenesis to anti-angiogenesis; 0 to 1; 'on' to shades of 'off'). It is a very tangled web - but all pieces are crucially informative to the entire active picture, per tissue (even cell), per instance. E.g., If the anti-angiogenic isoform of VEGF seems to predominate at the mRNA level, then one might assume vessel formation is being disallowed at the protein level. So, as might be expected, ultimately, only tangible protein analysis will help spell the truth nonetheless; since expressed/translated functional protein (as far as modern dogma still largely implies) is the final, intended poetry of the DNA code anyway. Gamble the pieces carefully, and interpret away.
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