Isolation of total RNA from 3 to 10 million cells (about 1 ml of cells).

1. Dislodge cells from the surface of the dish by triteration or sharp tapping of the containiner.  Mix 25 ul of cell suspension with 25 ul of trypan blue and load about 20 ul into the hemocytometer.  Count the number of cells in the central 9 squares.  The cell density = number of cells x 2 x 10,000 cells/ml. This accounts for the 2-fold dilution into trypan blue.  Note that blue cells are dead.  If there are a lot, something is wrong.

2. Collect the remaining cells in a 1.5 ml tube by centrifuging 3000 rpm for 3'.  You might need to do two spins in the same tube if the volume of the media exceeds the tube.

3. Add 100 ul of Trizol Reagent and disperse the cells by triteration.

4. Incubate 5 minutes at room temp.

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

6. Microfuge 12,000 x g at 4 ^oC for 15 min.

7. Transfer upper aqueous phase (contains RNA) to a fresh tube.  Avoid transferring the white interface which contains DNA.

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

9. Microfuge at 12,000 x g 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 12,000 x g at 4 ^oC for 5 min. and discarding supernatant.  Repeat the wash a second time.

11. Air dry RNA pellet.

12. Dissolve in 50 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 1000 ul of DEPC water.  1 OD₂₆₀ = 40 ug/ml.  Recovery will be 50 to 100 ug of RNA.  Adjust final concentration to 1 ug/ul.

• 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. (The following describes reaction conditions for 1 sample treated with RT.  A "no RT" control can be done to control for contaminating DNA.  This reaction is done the same except DEPC water substitutes for RT.)

1. Combine the following in a PCR tube:

• 50 to 250 ng of total RNA
• 1 pmole of RP49-B primer*
• 1 pmole of first primer for desired RNA
  
• 0.5 ul of 10 mM dNTP
• DEPC water to a final volume of 6 ul

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

3. Premix the following on ice and add to the RNA sample on ice:

• 2 ul of 5X first strand buffer (Gibco/BRL: 250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl₂)
• 1 ul 0.1 M DTT
  
• 0.75 ul of DEPC water (or enough to give a final volume of 4 ul)
  
• 0.05 ul 40 U/ul RNasin (optional, based on USB catalog)
• 0.2 ul (40U) M-MLV reverse transcriptase

4. Incubate the 10 ul sample at 37 ^oC for 50 minutes, then 95 ^oC for 5 minutes.

5. Store cDNA (and no RT) control at -80 ^oC.

PCR amplification with Pfu polymerase

1. Prepare the following mixture on ice.

• Water so the final volume of the reaction is 25 ul
• 5 ul of 5X Vent buffer (from our LM-PCR protocol)
• 12.5 pmoles of RP49A
• 12.5 pmoles of RP49B
• 12.5 pmoles of each primer for the target mRNA
• 1 ul of cDNA
• 0.2 ul of Pfu from 5U/ul stock

2. Preheat PCR block to 95 ^oC. Transfer samples from ice to preheated block and subject to the following temperature regimen:

• 95^oC for 3 min.
• 55^oC for 1 min.
  
• 72^oC for 1 min.
  
• 19 cycles of 95^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 1.5% 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. 

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PCR amplification with Gene Choice Taq

1. Prepare the following mixture on ice.

• Water so a final volume of the reaction is 25 ul
• 2.5 ul of 10X Gene Choice Buffer
• 0.5 ul of 10 mM dNTP
• 5 pmoles RP49A
• 5 pmoles RP49B
• 5 pmoles of each primer for the target mRNA
• 1 ul of cDNA
• 0.2 ul of Gene Choice Taq (5U/ul stock)

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

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Analyze 10 ul of PCR product on a 1.5% 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. 

*RP49 turns out to be an excellent internal control.  We have the following pair of primers: