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

446 Gilchrist and Befus
Fig. 1. Schematic representation of the steps involved in the general protocol outlined in
the text. After fixation of the cells in suspension, the crosslinks that formed during fixation are
reduced by limited protease digestion. To remove the possibility of amplifying DNA during
the PCR, a DNase step is then performed to remove contaminating genomic DNA. A reverse
transcription reaction is then performed to convert the mRNA to cDNA. PCR amplification with
gene specific primers is then completed. The PCR product is then detected by chromogenic,
fluorescent or radioactive means. Many commonly used RT in situ PCR procedures follow
similar guidelines, differing in specific details, such as fixative used, protease used, digestion
time, and method of detection.
experiences. The inclusion of appropriate controls in every run helps insure accurate
results. Controls to indicate that genomic DNA has been removed as a source of
false-positive signals (negative control), or failure of PCR amplification because
of inadequate protease digestion or flaws in the RT-PCR protocol resulting in falsenegative
results (positive control) must be tested on every slide. Therefore, a slide
schematic of the necessary controls and test spots is included to aid in the elimination
of spurious results (Fig. 2).
2. Materials
1. Phosphate-buffered saline (PBS): 130 mM NaCl, 10 mM sodium phosphate, pH 7.4. Store
at room temperature.
2. 10% neutral buffered formalin (BDH). Store at room temperature.
3. Heparinase I (for heparin containing mast cell populations; Sigma).
4. Heparinase buffer: 5 mM Tris-HCl, pH 7.5, 1 mM CaCl 2 , 7.5 U RNasin.
5. Pepsin (5000 U/mL), made fresh in 0.01 N HCl for immediate use (Boehringer
Mannheim).
6. RNase-free DNase I (Boehringer Mannheim).
7. DNase solution: DNase I (10 U/µL) in 0.1 M sodium acetate, pH 5.0, 5 mM MgSO 4 .