Reverse Transcriptase Polymerase Chain Reaction

1. In a microcentrifuge tube, adjust the volume of 1 ig total RNA up to 12.9 iL with

DEPC-treated water.

2. To the reaction tube, add 1 iL (0.5 ig) of driving primer [oligo(dT)] and mix gently.

3. Incubate at 70°C for 10 min, and then chill on ice for 1 min.

4. To the reaction mix, add the following, in order:

- Glass plate

3-5 cm slack of paper towels

- Glass plate

3-5 cm slack of paper towels

3 pieces dry paper Hybond N'Membrane

Dish filled with 20X SSC

Fig. 1. Schematic representation of Northern blot transfer apparatus.

5X first-strand buffer RNase inhibitor (40 U/iL) NTP mix (25 mM) MMLV-RT (200 U/iL)

Mix gently.

5. Incubate the reaction mix at room temperature for 10 min, followed by 1 h at 37°C.

6. Heat inactivate the enzyme at 90°C for 5 min and then chill on ice for 10 min (see Note 17).

7. Set up the PCR reaction as follows:

1 iL reverse transcription mix

5 iL 10X PCR buffer

0.5 iL Taq DNA polymerase (5U/iL)

to a final volume of 50 iL with RNase-free distilled water.

8. Overlay the reaction mix with mineral oil. Run the PCR reaction for an initial denaturation step at 95°C for 10 min, followed by 35 cycles (denaturation for 15 s at 95°C, hybridize primers at 60°C for 40 s, primer extension at 72°C for 2 min).

9. Transfer the lower layer to a fresh microcentrifuge tube (see Note 18).

10. Products can be visualized by agarose gel electrophoresis.

11. This protocol is not appropriate for quantitative RT-PCR (see Note 19).

1. The lipofectamine reagent should be vortexed before adding to the solution.

2. The DNA and lipofectamine ratios can vary for a variety of cell types and the amount of lipofectamine and DNA required to give optimal transfection efficiency can be titrated. The values suggested here are for standard transfection of b-cell lines such as MIN6 (5) and b-TC (6).

3. This should be optimized for the cells in question; however, 5 h is a good starting point.

4. This method describes measuring luciferase activity by performing a standard luciferase assay; however, luciferase expression can also be measured by single-cell imaging techniques (7).

5. Samples can be frozen at -20°C and assayed at a later stage.

6. Before placing the tube in the luminometer, wipe the tube with ethanol to reduce interference. Also, store tubes out of direct sunlight, as this can interfere with measurements.

7. The value obtained should be in the region of 100, and if it is much higher, the lumi-nometer should be washed through again with distilled water.

8. The formaldehyde should be added and the gel should be poured in a fume hood.

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