In Situ RTPCR

3.4.1. DNase Treatment

1. Prepare a RNase-free, DNase solution and add 10 pL of solution to each well.

2. Incubate the slides overnight at 37°C in a humidified chamber. When using liver tissue, this incubation should be extended an additional 18 to 24 h.

3. After incubation, rinse the slides with a similar buffer solution that was prepared without the DNase enzyme.

4. Wash the slides twice with DEPC-treated water.

It is advisable to use a premade RT reaction solution that contains all the components necessary to carry out the reaction; one only has to add the mRNA template and a primer (see Note 9). However, if one desires to prepare his or her own reaction solution, then use the RT reaction solution described in Subheading 2., step 27.

1. Add 10 pL of cocktail to each well. Carefully place the cover slip on top of the slide (see Note 10).

2. Incubate at 42°C or 37°C for 1 h in a humidified atmosphere.

3. Incubate slides at 92°C for 2 min.

4. Remove cover slip and wash twice with distilled water.

5. Proceed with the amplification procedure, which is the same for both DNA- and

RNA-based protocols (see Notes 11-13).

3.4.3. Amplification Protocols (Protocol for Use With Conventional Sealing Technologies (Such as Frame-Seal Incubation Chambers)

1. Prepare an amplification cocktail containing the following: 1.25 |iM of each primer (see Note 14), 200 |M dATP, dCTP, dGTP, dTTP, 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl, 1.5 mM MgCl2, 0.001% stabilizer (including BSA and gelatin); and 2.5 U of DNA polymerase (see Note 15).

2. Layer 8 |L of amplification solution onto each well with a P20 micropipet so that the whole surface of the well is covered with the solution.

3. If using a single-well slide for a tissue section, add 12-20 |L of the solution to the well.

4. In all cases, be careful—do not touch the surface of the slide with the tip of the pipet.

5. Seal the slides as described herein (Fig. 1):

Place the plastic cover slip on top of the slide, or if using tissue sections, use a second slide instead of a cover slip.

Carefully seal the edge of the cover slip to the slide.

6. Place on a heat block for 90 s at 92°C to further cure the adhesive.

7. Place slides in an appropriate thermal cycler.

8. Run 30 cycles of the following amplification protocol: 94°C, 2 to 3 min; 45 to 55°C, 1 to 2 min; 72°C, 1 to 5 min depending on the size of amplicons (see Notes 16-18).

9. After the thermal cycling is complete, open the slides.

10. Carefully, pry off the plastic cover slip. For other sealing technologies, follow the manufacturer's instructions.

11. Place the opened slides on a heat-block at 92°C for 1 min. This treatment helps immobilize the intracellular signals.

12. Wash slides with 2X SSC at room temperature for 5 min, two times.

13. The amplification protocol is now complete and one can proceed to the in situ hybridization procedures (see Note 19).

3.4.4. One-Step RT Amplification

1. If one uses RT enzymes to manufacture complementary (c)DNA, which is subsequently amplified by any of the suitable thermostable RT/DNA polymerases (that both RT and DNA capacity) like rTth, use the typical cocktail described in Subheading 2, step 28 for this single-step reaction.

2. This reaction requires a slightly variant thermal cycling profile. Our laboratory uses the following amplification protocol: 42°C, 45 min; 92°C, 3 min; 42°C, 15 min; 92°C, 3 min; 42°C, 15 min; and then, 29 cycles of the following profile: 93°C, 1 min; 53°C, 1 min; 72°C, 1 min.

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