To maximize cDNA synthesis yield, reverse transcription was performed twice with 6 μg of purified RNA for each replicate (total of 12 μg/replicate) using random hexamer primers (Invitrogen) and Superscript II reverse transcriptase (Invitrogen).
The experimental design at the site included a randomized complete block with four replicates (a total of 12 experimental plots: 3 treatments × 4 replicates).
As stated above, we run the model for each of the 4 frequencies (900 Hz, 3, 12 and 25 KHz) at each of the 7 altitudes and each of the 5 replicates, a total of 140 runs.
With 5 technical replicates, a total of 54 microarrays were analyzed.
With four different conditions each with two biological replicates a total of 8 samples were analyzed.
The experiment was organized as a randomized complete block design, with 6 treatments and 15 replicates (blocks), with 10 palms per replicate (a total of 150 palms per treatment).
This experiment was repeated twice, with three replicate samples per assay, resulting in two biological replicates and a total of six replicates per sample.
For each biological replicate, a total of eighteen seedlings were grown (six seedlings each in three pots).
For each treatment, qRT-PCR was performed on one randomly chosen RNA sample used in the array experiments, along with two independent replicates, for a total of three replicate RNA samples for each treatment.
Quantitative reproducibility was instead expressed as Pearson correlation coefficients (r, dot plots), which were calculated based on the abundance values obtained for each protein or peptide in two different replicates, for a total of three replicate combinations per method.
