Materials
A laboratory equipped with all machinery and chemicals needed to carry
out the procedures.
Methodology
We used two different approaches; both were different types of transcriptome profiling techniques. The first has been published only once before and not in an international paper. The second approach is a well established technique.
Approach 1 (cDNA-RAPD; Jiang et al., 1999)
1. Expose two weeks old wheat plants at 4 degrees Celsius in a growth
chamber and take leaf samples every 15 minutes up to an hour.
2. Extract total RNA from leaf samples, followed by isolation of mRNA.
3. Reverse transcribe the mRNA to synthesize cDNA.
4. Run the RAPD (Random Amplified Polymorphic DNA) procedure with the
cDNA with primers RC2 through RC29.
5. Run an agarose gel in electrophoresis, stain with ethidium bromide
and photograph under UV light.
Approach 2 (cDNA-AFLP; Bachem et al., )
1. Cut cDNA with restriction enzymes EcoRI and MseI.
2. Ligate EcoRI and MseI adaptors to cut cDNA.
3. Pre-amplify the adaptor-ligated cDNA with primers complementary to
adaptors using PCR.
4. Perform selective amplification with primer pairs complementary to
adaptors+ three variable bases.
5. Run agarose gel electrophoresis.
6. Take picture of gels.
7. Analyze the gel profiles.