The extraction procedure used was the same as described above, but with the exception that 15 cycles (1 1.5 h) were used in the extraction step instead of a specific extraction time (1 h).
RNA isolation from tumour tissue isolated by manual microdissection and cDNA preparation steps were accomplished as described previously (Kuramochi et al, 2006), with a slight modification in the extraction step using RNeasy Mini Elute spin-columns (Qiagen, Chatsworth, GA, USA).
For the extraction step, we used the Soxhlet extractor Gerhart 173200 EV (Gerhart, Königswinter, Germany) with a cellulose extraction thimble (Whatman, Brentford, UK).
RNA was isolated from tumor tissue and cDNA was prepared as described previously [ 12], with a slight modification to the extraction step, which used RNeasy MinElute spin columns (Qiagen, Chatsworth, GA, USA).
The extraction step was optimized using fractional factorial and central composite designs including the following experimental factors: saline concentration; extraction time; desorption time; agitation velocity; headspace volume.
The extraction step was performed using 10 mL acetonitrile.
The extraction step was optimised by using an experimental design.
The effects of various experimental parameters in the extraction step were also studied using response surface methodology.
Estrone, estradiol, androstenedione, testosterone, DHEA and DHEAS were assayed at the Quest Diagnostics Nichols Institute (San Juan Capistrano, CA, USA) using an extraction step (except for DHEAS) followed by RIA.
Extraction of polar metabolites from brain tissue was carried out using the two-step extraction method described previously [ 75], using methanol, water and chloroform for the optimal extraction of polar metabolites.
