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Cross-contamination
Selection of Primers
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PCR is a very sensitive technique and extreme caution should be taken to avoid cross-contamination of samples.
The first step in RT-PCR is to select primers and make complementary DNA (cDNA). This is best synthesised using random hexanucleotide primers, rather than virus-specific ones. This increases the sensitivity and enables the same copy DNA product to be subsequently amplified by PCR using several sets of specially designed primers.
Procedure for reverse transcription:
Dissolve extracted tissue RNA pellet in RNAse-free DEPC-treated water at 1 mg/ml. In the case of RNA derived from PBMCs use 1/4 to 1/10 of the RNA extracted from 10 ml of whole blood. Use sterile thin-walled 0.75 ml microcentrifuge tubes specially manufactured for use in thermocyclers.
1. Prepare RNA for reverse transcription:
5 µl of RNA solution (approximately = 5 µg)
2 µl of random hexanucleotide primers (50 ng/µl)
3 µl of DEPC-treated water
10 µl total
Incubate for 5 min at 70°C to disrupt any RNA secondary structure and cool at room temp for 10 min to allow primer annealing. Centrifuge briefly (10-20 s) before opening to ensure all of the sample is at the bottom of the tube.
2. Prepare RT buffer mix
Final concentrations in reaction
4 µl 5x RT buffer 50 mM TRIS-HCl pH 8.3
3 mM MgCl2
75 mM KCl
2 µl BSA (acetylated, 1 mg/ml) 0.1 mg/ml
2 µl DTT (0.1 M) 10 mM
1 µl dNTPs (10 mM each) 0.5 mM each dNTP
1 µl reverse transcriptase 200 units
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10µl total
Note: Make enough mix for one extra tube and add 10 µl of this mix to each RNA sample.
Mix gently and centrifuge (10-20 s) briefly to ensure all the reactants are at the bottom of the tube. Incubate at room temperature for 5 min and then at 37°C for 30-60 min. If necessary the cDNA product can be stored at -20°C until used for PCR.
Procedure for PCR amplification:
Take 5 µl of the cDNA product and add:
5.0 µl 10x PCR buffer
1.5 µl 50 mM MgCl2
35.0 µl Sterile distilled water
0.5 µl Taq - polymerase (5 U/ul)
1 µl dNTPs (10 mM each)
1 µl (10 pmol) Primer 1
1 µl (10 pmol) Primer 2
To make the final reaction solution 1.5 mM, add 1.5 µl of 50 mM MgCl2 to each tube. Mix, briefly (10-20 s) centrifuge the mixture and overlay with 50 µl mineral oil.
Note: overlaying with mineral oil is not required if the thermocycler has a heated lid to prevent evaporation.
Place tubes in PCR machine and select the appropriate programme.
Setting up the thermocycler:
Any make of thermocycler may be used but the results may vary depending on the machine. Different time settings can be tried to obtain the optimum results, but the following cycles have been found to work satisfactorily on several different types of commercial thermocycler.
Programming the machine -
Hot start:
Step 1: temp 94°C time 5 min
Denaturation:
Step 2: temp 94°C time 1 min
Annealing:
Step 3: temp 55°C time 1 min
Synthesis:
Step 4: temp 72°C time 2 min
Repeat steps 2 to 4 for 30 cycles
Extension:
Step 5: temp 72°C time 7 min
Hold temperature*:
Step 6: temp 4°C time hold
*: optional since some machines are not designed to refrigerate and DNA can be left overnight at room temperature if necessary.
Analysis of RT-PCR products:
An aliquot of each reaction (usually 8 µl) is taken, mixed with one quarter volume of 5x gel loading buffer, and loaded and run on a 1.5% agarose gel. Suitable markers, either 123 or 100 base pair ladders, should always be used.
Optimising the reactions:
Since PCR is completely dependent on the primers annealing (binding) to their template cDNA, and the kinetics of this process are critically dependent on the base composition and sequence of the primers being used, the optimal conditions of salt concentration and temperature for PCR will vary with different primer sequences. Factors such as the Mg2+ concentration and the annealing temperature can greatly affect the efficiency of the PCR reaction and the production of non-specific DNA products. Commercial kits are available to optimise PCR reactions.
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