D. Analysis of macrophage foam cell gene expression by real-time quantitative RT-PCR.
Real-time quantitative RT-PCR is a very sensitive method which allows for measurements of low abundance transcripts and, unlike Northern or RNase protection assays, requires only a very small amount of total RNA (typically 100 pg - 1ng). A comprehensive review of the methodology has been done by Bustin, 2000. RT-PCR and subsequent PCR are both carried out in a single sealed optical tube using gene specific primers and fluorogenic probes.
1. Prepare a master mix containing the following for each reaction: 1X first strand buffer (50mM Tris-HCl pH 8.3, 75mM KCl, 3mM MgCl2), 5mM DTT, 0.3mM dNTP mix, 20U SuperScript II reverse transcriptase enzyme, 2.5U Taq DNA polymerase, 40U RnaseOut RNase inhibitor,0.625 g acetylated BSA, and 1X 5-carboxy-X-rhodamine internal reference dye (Invitrogen Life Technologies, Carlsbad, CA) in optical tubes.
2. Add 50nM of the forward primer and reverse primer, and 100nM of the probe to the master mix for each reaction.
3. To 5 l of samples (100 pg) and appropriate standard RNA (10 ng - 1 pg serial dilutions), add separately 20l of the master mix (see Special Considerations, Note 4).
4. Set the Sequence Detection System
7700 to the following cycling
conditions: RT-PCR stage (95C,
10 sec; 45C, 50 min; 95C, 2min)
immediately followed by 40 cycles of
PCR amplification (denaturation:
95C, 15 sec; annealing/extension: