I am trying to design an experiment about TLR7 and TLR8. I have a problem with the RT-PCR standards. I know I have to get standards and dilution process but where can I find standards for real time? Should I use Applied Bio. Step one cycler?
The easiest way is to clone out your gene into a generic plasmid and do a miniprep of it. Then you calculate the plasmid length/molarity. This will give you plasmid copies per ml. Then just add a known amount of plasmid per well for your standards. I usually start at 10^8 copies and do 10 fold dilutions down to 10. This way you can make one master mix for the whole plate and you take care of primer efficiencies since it is the same gene. FYI the ABI step one is a fine machine and will do great. Are you using SYBR or Taqman chemistry? It does not really matter for your standards but only for multiplexing wells, SYBR can't, Taqman can.
Thank you for your response. I am going to use the taqman. Also I am going to use cDNA, is this have the same principle with the plasmid?
It does not matter what sample/unknown you are going to run. What you want for your standard is an absolute number. Using a plasmid for a standard allows you to get a very precise number because you have more control over your variables. If you use genomic material you have to estimate genome size and that can be off quite a bit due to shearing and such.
If you are wanting to do a ratio of expression of gene of interest to housekeeping genes that is a whole offer set of problems. But sometimes that is the beeter option when your experiment calls for it.
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