RT-PCR analysis
Semiquantitative (competitive) PCR analysis was carried out for 38 cycles (annealing temperature 63°C) using as template cDNA (8 ng) obtained by priming poly(A)+ mRNA with an iaaM specific primer (5'-AATAGCTGCCTATGCCTCCCGTCAT-3'). The mRNA was extracted from either young flower buds (5,8,11 mm) or eggplant fruits (placental tissue from fruits either 40 or 280 mm long). As an internal standard, 0.5 fg of a 600 bp long DefH9 cDNA fragment was used in the PCR assay. To amplify the 161 bp long amplicon an iaaM specific primer (5'-GGGTGAATTAAAATGGTCATACAT-3') and a DefH9 specific primer (5'-CTTTGGAACTCGTGTTGAGCTCTCA-3') were used. For the internal standard, a 3' primer (5'-TGAGCATTGATCTCCTGAGTGGTGT-3') together with the DefH9 specific primer were used to produce the 351 bp long amplicon. The PCR assays were performed with a thermostable DNA polymerase mixture (Expand High Fidelity PCR system, Roche) in presence of 3 μCi of 32P dCTP. The intensity of the bands was quantified by using an Instant Imager (Packard, Meriden, CT).