6 protocols
The average fluorescence intensity for each fluor and for each gene was determined using the ImaGene program (BioDiscovery Inc., Los Angeles, CA). Median background fluorescence around each gene spot was calculated and subtracted from each spot. Signal values <500 (two to three times the average background stdev) were manually raised to 500 to avoid extreme expression ratios. Subgrid Loess normalization was performed using the limma library of the R statistical software
Slides are scanned at variable laser power and PMT voltage so that the number of saturated spots is below 1% and so that spots in the Cy3 and Cy5 channels have the same average intensity
(Parameters: Scanning hardware = ScanArray [PerkinElmer], Scanning software = Scanning software)
- Mix in a a 200 µl PCR tube:

49.8 µl labeled probe

10.5 µl 20X SSC

7 µl Yeast tRNA (2 µg/µl)

2.7 µl 10% SDS

Total 70 µl

- Place a microarray in the hybridization chamber

- Add 12.5 µl of 3X SSC to each groove in the hybridization chamber1

- Heat probe for 1 min, 95°C

- Spin 1 min, max speed

- Rapidly add probe to microarray, lay cover slip slowly on top of solution2

- Close lid, immerge chamber into 64°C water bath. WORK QUICKLY.

- Hybridize for 16 hrs without agitation.

(Parameters: Chamber type = OTHER: TeleChem hyb chamber, Quantity of label target used = 2, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume = 70, Volume unit = Micro litre, temperature = 64)
Total RNA was prepared from leaves by grinding in liquid N2, followed by phenol/chloroform extraction and two LiCl and sodium acetate precipitations.

PolyA RNA was prpared with Quiagen oligotex resin
(Parameters: Extracted product = polyA_RNA, Amplification = none)
- Add to two 200 µl PCR tube:

2 µl oligo-dT (21 mer, 1µg/µl)

x µl mRNA (2 µg) from control or test sample

y µl H2O (final volume = 13.4 µl)

- Heat 5 min, 70°C (PCR machine).

- Leave 5 min at RT.

- Add to each tube:

6 µl 5X SuperScript II buffer

3 µl 0.1 M DTT

3 µl Cy3-dCTP (1 mM) or Cy5-dCTP (1 mM) (Amersham)

0.6 µl dNTPs (25 mM dATP, dTTP, dGTP; 10 mM dCTP)

2 µl SuperScript II Reverse Transcriptase (BRL)

2 µl RNAse Inhibitor (15 U/µl, BRL)

(When labeling more than one sample, prepare a mix of the above compounds)

- Mix well and incubate at 42°C for 1-2 hr (PCR machine)

- Pool the two tubes (control + test sample)

- Add: 2.65 µl 25 mM EDTA

3.3 µl 1 M NaOH

- Incubate 10 min, 65°C (PCR machine)

- Add: 3.3 µl 1 M HCl

5 µl 1 M Tris pH 6.8

- Purify probe with Qiagen MinElute PCR purification kit and elute with 10 µl

(Parameters: Amount of nucleic acid labeled = 2, Mass unit = Micro gram, Label used = Cy5, Amplification = none)
growth protocol
Transcriptome of unchallenged three-week-old Arabidopsis plants in controlled condition (no treatments) is analysed in wild-type and mutant plants