Sybr RT PCR problem - (Aug/29/2005 )
This may well be an utterly stupid problem, but ive only just started doing RT PCR and dont know if there im doing something obviously wrong.
ive designed some sybr primers to all the specs that you are supposed to, to give me an intron/exon boundary transcript of 150bp (im looking at primary transcript levels to try and assess transcription). and ive done the matrix of different primer concentrations like you are supposed to.
i get Ct values in the low 20's which makes me think i have transcript. However there is no increase in transcript from the unstimulated to the stimulated... which there should be, as the control primer/probe for the same gene shows induction on stimulation in the exact same samples.
there is no contamination by genomic dna as i run a RNA -RT control too.
im multiplexing my samples with GapDH: is this the problem? or is it something else? i dont understand!!!
It may help you to know that you can't multiplex with Sybr, as any increase in the reaction will fluoresce, you can't distinguish between your sample and (in your case) the control causing the increase. To multiplex you need to use specific probes with different fluorescence emission/excitation maxima so that the different components in the reaction canbe distinguished. Taqman does this I think.
im using gapdh primer:probes labelled with vic to act as the control which seems to respond fine. and give decent ct values.
Ah i understand now.
even though the GAPDH has a vic probe, the GAPDH product will soak up the Sybr and drown out all the target genes amplification.
well at least i know my samples are pretty similar in DNA concentration!
all is not lost! phew!
Did you solve your problem already?
I have attached a link to the Invitrogen home page, where you can get much information on primer/probe labeling, fluorescence crosstalk effects and so on. I work for a company, that develops diagnostic real-time PCR Kits, I know what I´m talking about . You can get many hints there.
spectra viewer: a tool for checking your colours:
and the Invitrogen molecular probes homepage: