I am studying the expression of a group of homologous genes and I need to check their expression by qRT-PCR. However, since these genes are highly similar, it is difficult to design primers that both differ them clearly and match the requirements of qPCR. For many of them
Does anyone know how can I check the expression of each individual clearly? Thank you.
Did you think of:
a) a hydrolysis probe in combination with MGB? Than you can design short probes that are focused on the small differences between those genes. In many qPCR systems you can use 5 different fluorochromes in different channels. In that way you can disciminate 4 or 5 different gene variants. One or two channels must be reserved for your reference gene(s)
b) dual probes, that bind at both sides at a single base difference and then perfrom hig resolution meting curve analysis. Such techniques are used to detect SNP's. Think of refenrence genes too!
c) Perform a reverse line blot test. In that way e.g. many subtypes of very related HPV genotypes were discriminated.
You may consider nested PCR. First amplify the whole set with universal primers and than run a second PCR that is specific for the different homologous genes. As you have enriched you genes of interest you may get away with less optimal primers.
Depending on availablilty of faclities and budget you could go for RNAseq, though less quantitative than qRT-PCR
Did you think of:
a) a hydrolysis probe in combination with MGB? Than you can design short probes that are focused on the small differences between those genes. In many qPCR systems you can use 5 different fluorochromes in different channels. In that way you can disciminate 4 or 5 different gene variants. One or two channels must be reserved for your reference gene(s)
b) dual probes, that bind at both sides at a single base difference and then perfrom hig resolution meting curve analysis. Such techniques are used to detect SNP's. Think of refenrence genes too!
c) Perform a reverse line blot test. In that way e.g. many subtypes of very related HPV genotypes were discriminated.
In any gene expression analysis, protein expression is a better read out than mRNA expression. If that would suit your study, then you could consider quantitative Western Blot assays.
If the genes are very similar, design a common reverse primer in a conserved region and anchor the specific forward primers upstream of the start codon between transcription starting site and translation starting site. Usually you can safely go 100 bp upstream of ATG.
In addition to all of the suggestions above, you may want to consider using specific internal probes that may be easier to design, and will yield more accurate results anyway. Look up "TaqMan probes".
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