John M. S. Bartlett.pdf - Bio-Nica.info

RT In Situ PCR 449
3. Wash slides in DEPC treated water for 1 min.
4. Then wash the slides in 100% ethanol for 1 min.
5. Allow the slides to air dry at room temperature.
3.6. PCR
1. Place 50 µL of the PCR master mix on EACH spot (see Note 9).
2. A good starting program for the TNF primers is outlined: 94°C for 2 min, followed by
30 cycles of: 94°C for 1 min , 45°C for 2 min, 72°C for 2 min, and a final step at 4°C
until ready to develop the slides. However, this may require optimization for each gene
and cell type studied (see Note 10).
3. Once the slides have finished cycling, remove the coverslip and transfer the slides to
wash buffer 1 for 5 min.
4. Do not allow the slides to air dry from this point on.
3.7. Digoxigenin Detection and Color Development
This step uses an alkaline phosphatase (AP)-labeled anti-digoxigenin antibody to
detect the incorporated digoxigenin-dUTP. Color development is accomplished with
NBT/BCIP Chromagen, which is oxidized to a purple/blue color by AP.
1. Prepare a 1300 dilution (see Note 11) of anti-DIG antibody using wash buffer 1 as the
diluent. Add 150 µL of the diluted antibody to the slide. Incubate the slides in a moist
chamber for 30 min at room temperature.
2. Wash the slides with wash buffer 2 for 5 min.
3. Prepare the substrate solution.
4. Add the substrate solution to the slides and develop for 5 min to 1 h at room temperature.
Monitor the purple/blue color development under the microscope.
5. Stop the color reaction by washing the slides in water.
6. Wash the slides for 1 min in 100% ethanol.
7. Wash in xylene, 1 min.
8. Mount using Permount and a coverslip.
3.8. Controls and Expected Results
The positive control (–DNAse, –RT, +PCR) should show intense nuclear staining in
>90% of the cell population. This indicates incorporation of digoxigenin into strand
breaks of genomic DNA by Taq polymerase and ensures that the protease digestion
and PCR were adequate (Fig. 3b).
The negative control (+ DNAse, –RT, +PCR) should show no staining. This indicates
that all genomic DNA has been digested and thus any staining in the test is caused
by mRNA expression (Fig. 3c).
The test (+ DNAse, +RT, +PCR), if positive, should show staining predominantly
over the cytoplasm and not in the nucleus. The cytoplasm is the correct cellular
compartment for most mRNA (Fig. 3a), although it may have an intense perinuclear
localization, especially in highly granulated cells (8).
3.9. Future Directions
Initial development of RT in situ PCR lent itself immediately to areas of pathology
and detection of viruses important in human disease. Publications in the field provided
new insights into the pathogenesis and involvement of viruses, such as human papilloma
virus (HPV) in cervical carcinoma (10) and Human Immunodeficiency Virus (HIV)