Growth conditions, shak ing and cell density were carefully controlled over time until harvesting. http://www.selleckchem.com/products/Calcitriol-(Rocaltrol).html Typically, cells were grown overnight in a culture tube containing 3 ml YPD. The overnight cul tures were then re inoculated in fresh media to OD600 0. 1 in 100 ml flasks containing 30 ml YPD with or without 10 uM FTase inhibitor I or 10 uM GGTI 298 and harvested at OD600 0. 8 for RNA extraction. RNA extraction, cDNA labelling and micro array hybridisation Total RNA, labelling and cDNA hybridization onto the microarray was performed as described by the array manufacturer and in Additional file 1. Briefly, RNA extraction was performed following the hot phenol method. Reverse transcription was per formed using Superscript II in the presence of either Cy3 or Cy5 dCTP and the manu factures standard conditions for the other nucleotides.

Dye swapping was used to minimise errors due to different incorporation capabilities of Cy3 and Cy5. At least three independent RNA extrac tions were performed for each pair of tests. Quantitative Real Time PCR To validate the array normalization and statistical analy sis for the FTI arrays we performed quantitative Real Time PCR. For this, ten genes were randomly chosen among the up and down regulated hits of FTase treated cells. As an endogenous control we used the YBR159W gene. Primers were designed using the Primer Express software from Applied Biosystems. The amplification efficiency of each pair of primers was tested using serial dilutions of genomic DNA as template and amplification under standard conditions. Efficiency was calculated as Eff 10 1 100.

All primer pairs had an efficiency between 90 110%, and showed a single peak in dissociation curves. Primer spe cificity was also controlled using agarose gel electro phoresis. Total RNA was isolated from yeast cells treated or not with FTase inhibitor I. The cells were in the exponential growth phase the drug was administrated at OD600 0. 2 and the RNA was extracted at OD600 0. 8. Reverse transcrip tion of 2 ug of DNase treated RNA was performed using random primers and Taqman Reverse Transcrip tion Reagents according to the manufacturers instructions. Amplification reactions were performed in a volume of 50 ul in the presence of SYBR Green PCR Master Mix, 300 nM Dacomitinib of each primer, and 2 ul of cDNA on an ABI Prism 7900 HT device. Amplification conditions were 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. Each amplifica tion was run in triplicate, and two independent experi ments were performed. Relative expression levels were calculated automatically selleck Imatinib Mesylate by the ABI Prism 7900 HT software with the Ct method using the YBR159W gene as the endogenous control.