Quantitative PCR

ABSTRACT

Quantitative PCR involves co-amplification of two templates: a constant amount of a preparation containing the desired target sequence and varying amounts of a reference template. After amplification, the concentration of the target sequence in the preparation of nucleic acid under test is established by interpolation into a standard curve. Quantitation of nucleic acids by PCR is best performed by real-time PCR. However, the following robust protocol, which uses radioactivity to quantify PCRproducts, remains useful when a real-time instrument is unavailable. The method can be easily adapted to other methods of quantification such as fluorometry.

MATERIALS

Reagents and Solutions

Chloroform dNTP solution (pH 8.0), containing all four deoxynucleotide triphosphates, each at a concentration of 20 mM MgCl2 (1 M) Placental RNase inhibitor (20 units/µl)

Enzymes and Buffers

Appropriate restriction enzymes and 10x buffers 　　Bacteriophage T4 DNA ligase and 10x buffer 　　Reverse transcriptase, required only if RNA is used as a template 　　Thermostable DNA polymerase and 10x amplification buffer as supplied by the manufacturer or homemade 　　500 mM KCl 　　100 mM Tris-Cl (pH 8.3, room temperature) 　　15 mM MgCl2

Nucleic Acids/Oligonucleotides 　

DNAmarkers for gel electrophoresis 　　Externally added reference (either DNA or RNA) of known concentration 　　Use a DNA reference to measure the concentration of DNA sequences and, if possible, an RNA 　　reference for RNA targets. A method to construct reference RNA is described in Protocol 15.2. 　　Sense and antisense primers, each 20 M in H2O 　　There is nothing unusual about the primers used in quantitative PCR. The standard rules for primer design apply. 　　Target nucleic acid 　　The target can be a preparation of DNA or RNA, either total or poly(A)+. Dissolve preparations of total RNA in H2O at a concentration of 0.5-1.0 mg/ml and preparations of poly(A)+ RNA at 10-100 　µg/ml. Dissolve DNA targets in 10 mM Tris-Cl (pH 7.6) at the following concentrations:mammalian genomic DNA, 100 µg/ml; yeast genomic DNA, 1 µg/ml; bacterial genomic DNA, 0.1 µg/ml; and plasmid DNA, 1-5 ng/ml.

Radiolabeled Compounds

[α-32]dCTP (sp. act. 3000 Ci/mmole at 10 mCi/ml) Gels/Loading Buffers 　　Polyacrylamide or agarose gel

Additional Items 　

Barrier tips for automatic pipettor 　　Fluorometer (optional; see Step 1) 　　Light mineral oil or wax bead (optional; see Step 5) 　　Materials for autoradiography or phosphorimaging 　　Microtiter plates or microfuge tubes, 0.5 ml and thin walled 　　Positive displacement pipette 　　Thermal cycler, programmed with desired amplification protocol 　　Water baths (94℃and, for RNA templates only, 75℃)

METHOD

Design and prepare a reference template suitable for the task at hand. Measure the concentration of the reference template as carefully as possible, preferably by fluorometry. Alternatively, estimate the amount of reference template after gel electrophoresis and ethidium bromide staining.

Make a series of tenfold dilutions (in H2O) containing concentrations of the reference template ranging from 10-6 to 10-12 M. After using the dilutions (Step 3), they should be stored at -70℃ for later use in Step 8.