Preparations
Prior to setting up real time PCR, you will need the following:1. Reagents and disposables from ABI including a real time PCR kit for either Sybr Green or Taqman. We use the Taqman universal PCR master mix about $2/ 50 ul reaction) which comes with 2x master mix to which you need only add DNA/RT, primers and probes.
Sybr green PCR reaction kit ABI 4309155 Taqman universal PCR master mix ABI 4304437 Optical plates DNA Facilty or Roche Optical sheets DNA Facility or Roche 2. Primers (Sybr green and Taqman) and probes (Taqman only). Probes should be 5' labeled with 6'FAM and 3' labeled with TAMRA. If ordering from Qiagen, they should automatically be HPLC purified
3. DNA or RT reactions (see RT protocol in DNA/RNA section of general protocols).
Setting up reactions
(The following reaction description will talk in terms of quantifying different genes relative to each other as for a gene expression study. However, the same description applies to species surveys where you want to discriminate different species using the same (or different) gene. In this case, gene in the following discussion refers to species.)
In setting up reactions it is important to use master mixes as much as possible. This reduces pipetting error (note: the Sybr green and Taqman master mixes do contain a ROX normalization dye which does correct somewhat for pipetting differences between the wells). We have been running these with each gene being one color though the Roche machine is a multicolor instrument. For this single color appraoch, each gene that you want to detect, you must set up a separate PCR reaction. This involves making a master mix for all the genes from a single sample that you wish to compare and then aliquoting them to separate tubes. You then make up a master mix of primers (or primer/probes for Taqman) and add then. It is useful to set up a grid with samples going across the rows and genes going down the columns. These need to be put in the special white qPCR plates available either from the DNA facility or from Roche.
The first time you test your primer/probes, it is useful to do a negative control (water) and then a dilution series of your positive control. This will confirm that you do not have contamination and that your are working in the exponential regime where critical cycle number is linear with template concentration. It is also useful to do have one cDNA sample where you did not add RT enzyme. This will test for genomic DNA contamination (which is always there unless you DNAse things).
The following is a sample set up for a Taqman study of six opsin genes. This might be a test case where the individual samples would be a dilution series of seven different concentrations (1x, 0.5x, 0.1x, 0.05x, 0.01x, 0.005x, 0.001x) to test linearity. A dilution series is handy because it also can give you information on the PCR efficiency of each gene (see data analysis). We often include a positive control with each set of unknowns which for cichlid opsins is the 6 gene construct. Using this in a dilution series will get all the efficiencies for all the genes.
We have now tested slightly smaller volumes and they work great:
For each of the 7 RT samples (concentrations), make up the following master mix: Reagent For 1 rxn For 6 genes (x6.5) 2x Taqman mix 10 ul 65 ul water 3.6 ul 23.4 RT reaction 0.4 ul 2.6 ul Total 14.0 ul 91 ul Mix well and spin. Add 15 ul to each tube in sample row. For each of the six genes, make up the following master mix: Reagent For 1 rxn For 8 RT samples Forward primer (3 uM) 2 ul 18 ul Reverse primer (3 uM) 2 ul 18 ul Probe (2 uM) 2 ul 18 ul Total 6 ul 54 ul Mix well and spin. Add 6 ul to each tube in gene column and pipette to mix.
This is the old way with slightly larger volumes:
For each of the 7 RT samples (concentrations), make up the following master mix: Reagent For 1 rxn For 6 genes (x6.5) 2x Taqman mix 12.5 ul 81.25 ul water 4.5 ul 29.25 RT reaction 0.5 ul 3.25 ul Total 17.5 ul 113.75 ul Mix well and spin. Add 17.5 ul to each tube in sample row. For each of the six genes, make up the following master mix: Reagent For 1 rxn For 8 RT samples Forward primer (3 uM) 2.5 ul 20 ul Reverse primer (3 uM) 2.5 ul 20 ul Probe (2 uM) 2.5 ul 20 ul Total 7.5 ul 60 ul Mix well and spin. Add 7.5 ul to each tube in gene column and pipette to mix.
Once the RT master mixes and primer/probe mixes are distributed, put on the optical sheet. Spin down the plate. You are ready to run the Roche Light Cycler. Make sure you signed up ahead of time for a 2.5 hr run.
07/04 KC