root - in vitro, standard medium, vertical petri dishes
Extracted molecule
total RNA
Extraction protocol
Pool of extract, 18:1ug, 16:1ug, 14:1ug.
Label
Cy5
Label protocol
labelling Cy3 and Cy5 indirect, amplification=yes, DNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
root - in vitro, standard medium, vertical petri dishes
Extracted molecule
total RNA
Extraction protocol
Pool of extract, 6:1ug, 4:1ug, 2:1ug.
Label
Cy3
Label protocol
labelling Cy3 and Cy5 indirect, amplification=yes, DNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
Hybridization protocol
7817 100(u)M Cy5 / col 100(u)M Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.
Scan protocol
GenePix Pro 3.0, Cy3:pmt voltage 532nm,650V,laser power 100%, Cy5:635nm,pmt voltage 650V,laser power 100%
Description
Study of 2 TOR mutants (7817 Strong, 7846 weak)
Data processing
The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.
