Isolation of total RNA from salivary glands.

1. Dissect 15 to 20 pairs of salivary glands transfer to 20 ul of PBS on ice.

2. Briefly spin glands to the bottom of the tube and remove 10 of supernatant.

3. Add 100 ul of Trizol Reagent.

4. Shake 5 minutes at room temp.

5. Add 20 ul chloroform, shake vigorously for 15 seconds, incubate at room temp. for 3 minutes.

6. Microfuge at 4 ^oC for 15 min.

7. Transfer upper aqueous phase (contains RNA) to a fresh tube.

8. Add 50 ul isopropanol to the aqueous phase, mix, and incubate at room temp. for 10'.

9. Microfuge at 4 ^oC for 10 min. (pellets RNA).

10. Remove supernatant.  Wash the pellet by adding 100 ul of cold 75% ethanol, vortexing briefly, microfuging at 4 ^oC for 5 min. and discarding supernatant.  Repeat the wash for a second time.

11. Air dry RNA pellet.

12. Dissolve in 10 ul of DEPC water.  Triterate and then incubate at 55 ^oC for 10 min.

13. Quantify RNA with the spectrophotomer by diluting 1 ul of RNA into 500 ul of DEPC water.  1 OD₂₆₀ = 40 ug/ml

• Note, you can also run a small amount of RNA on a 1% agarose gel (TBE buffer) and visualize the rRNA by ethidium bromide staining.

14. Store RNA at -80^oC.

cDNA synthesis (I've cut Mike's original volumes in half to provide a no RT-control).

1. Combine the following in a PCR tube:

• 100 to 500 ng of total RNA
• 2 pmoles of RP49-B primer
• 1 ul of 10 mM dNTP
• DEPC water to a final volume of 13 ul

2. Heat 65 ^oC for 5 min., chill on ice, spin down condensate and store on ice.

3. Add the following on ice:

• 4 ul of 5X first strand buffer (provided with the Superscript II)
• 2 ul 0.1 M DTT (also provided with the Superscript II)
• 1 ul 40 U/ul RNasin (optional)

4. Mix and divide the sample into 2 x 10 ul  portions.  Incubate each tube at 42 ^oC for 2 min. (one tube will be a no RT control)

5. Add 0.5 ul of Superscript II reverse transcriptase to one tube.  Incubate at 42 ^oC for 50 minutes, then 70 ^oC for 15 minutes.

6. Optional step (not necessary for RP49): add 2.5 ul of 5X RNase H buffer to each tube and 0.2 ul of RNase H.  Incubate 37 ^oC for 20 min.

7. Store cDNA and no RT control at -80 ^oC.

PCR amplification (note, it might be worth testing if this can be scaled down by a factor of 2).

1. Combine in a PCR tube on ice (note you can make a master mix with everything but the cDNA (or no RT control):

• Water so a final volume of the reaction is 50 ul
• 5 ul of 5X Gene Choice Buffer
• 3 ul of 25 mM MgCl₂
• 1 ul of 10 mM dNTP
• 1 ul of 10 uM RP49A
• 1 ul of 10 uM RP49B
  
• 0.4 ul of Gene Choice Taq (5U/ul stock)
• 2 ul of cDNA

2. Preheat PCR block to 94 ^oC. Transfer samples from ice to preheated block and subject to the following temperature regimen:

• 94^oC for 3 min.
• 55^oC for 1 min.
  
• 72^oC for 1 min.
  
• 19 cycles of 94^oC-1'/55^oC-1'/72^oC-1'
• 72^oC for 9 min.
• store at 10^oC

Analyze 10 ul of PCR product on a 2% Agarose gel.  The spliced RP49 message will give a PCR product of 220 bp and contaminating genomic DNA or unspliced RNA will give a PCR product of 286 bp.