experiment treated with 100ng/ml PGA (synthetic folic acid), third cell culture, first of the duplo
Extracted molecule
total RNA
Label
Cy5
Description
The 3DNA Array 350 HS Expression array detection kit for microarrays (Cy3/Cy5 Kit, Genisphere, Hatfield, USA) was used for both cDNA synthesis and microarray hybridization. Briefly, 5 µg of total RNA from each sample (4 x 3) was reverse transcribed in duplo using 5 pmol capture sequence primer. The Cy5 specific primer was used for the individual samples and the Cy3 specific primer was used for the reference samples, consisting of a pool of RNA extracted from all samples. In total, 4 x 3 x 2 = 24 reactions were performed for both Cy5 and Cy3 samples. For all individual reactions, total RNA (5 µg in 1-10 µl) and RT primer (1 µl) were brought to a final volume of 11 µl with nuclease free water and incubated at 80 degrees C. Afterwards, 1 µl of Superase-In was added and a reaction mix consisting of 4 µl 5x SuperScript II first strand buffer, 1 µl dNTP mix (10 mM each for dATP, dCTP, dGTp, dTTP), 2 µl 0.1 M DTT and 1 µl Superscript II enzyme (200 units) (Invitrogen). The reaction was incubated at 42 degrees C for 2 hours. The reaction was terminated by adding 3.5 µl 0.5 M NaOH/50 mM EDTA and incubation at 65 degrees C for 10 minutes. The reaction was neutralized with 5 µl 1 M Tris-HCl, pH 7.5. The Cy5 and Cy3 reactions were then combined per sample, purified with the Qiaquick PCR purification kit (Westburg) and precipitated overnight at -20 degrees C using 1/10 volume of 3 M sodium-acetate (pH 5.2) and 2.5 volumes of 96% ethanol.
Data processing
ArrayVision for visualisation of the array, GeneMathsXT for Rikilt normalisation and further data-analysis
