Below are the four steps that were followed, and a complete list of all the materials used in this project.

1. RNA Extraction From Wheat Leaves

Grind about 0.5g of leaf tissue in a -20° C pre-cooled      RNase-free mortars and pestles in liquid nitrogen.
Transfer to an RNase-free container containing 1ml of Trizol      per 50-100 mg of leaf tissue, transfer ground leaf and
     mix thoroughly.
Transfer 1 ml of extract to RNase-free 2ml tube.
Vortex vigorously and incubate at 15-30°C for 5 minutes.
Add 500 µl of chloroform to each tube a vortex for 15      seconds.
Centrifuge at 3000 rpm (rotations per minute) at 2-8°C for
     10 min.
Transfer colorless aqueous phase to new tube RNase-free      2ml tube.
Repeat chloroform extraction one more time.
Transfer supernatant to new RNase-free 2 ml tube.
Add 500 µl Isopropanol per 1 ml Trizol in initial      homogenization step.
Incubate at 15-30°C for 10 min.
Centrifuge at 3000 rpm at 2-8°C 10 min.
Discard supernatant.
Wash RNA pellet in 1 ml of 75% ethanol.
Centrifuge at 300 rpm at 2-8°C for 10 min.
Dry briefly and re-suspend in 100 µl DEPC-treated water.
Heat at 65°C to ensure complete re-suspension.
Pool aliquots from extractions and quantify RNA.


2. Synthesizing First Strand cDNA for Real-time PCR

Add 10X Buffer RQ1 (2 µl), DNase (5 µl), and H2O (up to 20      µl)
Incubate at 37°C for 30 min.
Add Reverse Primers 1& 2 (2 µM), 5X First Strand buffer
     (10 µl), DTT (5 µl), dNTPs (2.5 µl), Superscript III RT
     (1.0 µl), and H2O (1.5 µl)
Incubate at 50°C for 1 hour.
Store at -20°C
Collect sample


3. Real-Time PCR Set Up

Make a cocktail of H2O (10.25µl), Full Velocity (31.25µl), Forward Primer (7.5µl)
Add Reverse Primer (7.5µl), Reference dye (1.0µl), Template      (1.0µl)
Aliquot 11.5 µl into each of the 5 new tubes.
Add 1 µl of the 1/5 dilution solution.


4. Place the sample in the Real-Time PCR machine.

Real-Time PCR Machine