Gene expression analysis using glass chip (micro) arrays

Microarray analysis was performed as described in the following sections. All microarray slides were purchased from Clive and Vera Ramaciotti Centre for Gene Function Analysis, University of New South Wales.

3.3.3.1 cDNA synthesis for microarray analysis

First strand cDNA was prepared from phenol extracted RNA from yeast cell cultures, as described in section 3.3.1.1. A volume 18.7 µl (20-25 µg) of DNase-treated total RNA samples was mixed with 13.5 µl of Mix 1 {according Table 3.2 reagent master Mix 1 of 28.35 µl was prepared for one slide (control and test sample)} and placed into PCR machine. The PCR machine was programmed at 65°C for 5 min and 42°C for 2 hrs and 15 min and final 65°C for 20 min. Following 65°C for 5 and 42°C for 5 min incubation the PCR reaction was paused and 5.95 µl of Mix 2 (from Table 3.3) and 2 µl of Superscript II enzyme were added directly to each sample the cycle was continued at 42°C for 2 hrs 15 min. Following this 4 µl EDTA (50 mM, pH 8.0) and 2 µl NaOH (10 M) were added, and the PCR cycler was set to 65°C for 20 min to hydrolyse the RNA. The reaction mixtures were then neutralized with 4 µl acetic acid (5 M).

3.3.3.2 Purification and labeling of PCR products

PCR products were purified on QIAquick (QIAGEN) columns according the manufactures instructions. In brief, 150 µl PB buffer (phosphate buffer) was added to each PCR product and this was applied to the columns. An additional 150 µl PB buffer was used to rinse the tubes and this was also added to the columns. Columns were centrifuged at 3,185 g for 1 min, the flow through was discarded and a volume of 700 µl (70% v/v) ethanol was added to each column and incubated for 1 min. Following incubation, columns were centrifuged at 3,185 g for 1 min and the flow through was discarded (the washing step with 70% ethanol was repeated twice). Finally, columns were dried by centrifugation at 3,185 g for 1 min.

For elution of cDNA, columns were placed on fresh sterile microfuge tubes and 25 µl of DEPC-treated distilled water was added to each followed by incubation at room

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temperature for 5 min. Columns were then centrifuged at 3,185 g for 1 min. Again 10 µl of DEPC-treated distilled water was added to each column and incubated at room temperature for 5 min, followed by centrifugation at 3,185 g for 1 min. A final volume of about 35 µl was collected. The collected cDNA samples were centrifuged in a Speedivac at a low speed for ~20 min to reduce the volume to 2 – 5 µl. Following the speedivac step, 9 µl of NaHCO3 (0.1 M, pH 9) was added to each sample and mixed thoroughly by flicking it.

Following cDNA synthesis and purification, for indirect labeling aminoallyl-modified nucleotides (aminoally-dUTP)were used for the subsequent coupling to Cy3 and Cy5 fluorescent dyes. The fluorescent dyes, Cy3 and Cy5, that were used to label cDNA for microarray analysis were provided in pellet form by supplier (Amersham). Each dye was resuspended in 18 µL DMSO and aliquot of 2 µL were prepared into each eppendorf tubes, all aliquots were centrifuged in speedivac until dry pellets formed and stored at 40C until required. In darkness, 2 µl of DMSO stock solution was added to each Cy3 and Cy5 pellet, and resuspended by repetitive pipetting. In darkness 2 µl of Cy3 and Cy5 dyes were then added to corresponding tubes containing (2 – 5 µl PCR products plus 9 µL of NaHCO3. The samples were mixed thoroughly by repetitive pipetting. Note that control samples were labeled with Cy3 (green) and experimental samples with Cy5 (red). The samples were incubated at room temperature in darkness for 1 hr 30 min.

3.3.3.3 Washes of labelled cDNA

Labelled cDNA was washed in darkness to remove unincorporated dyes. This was carried out by adding 65 µl of PB buffer to each sample, mixing thoroughly by repetitive pipetting and loading onto QIAquick columns. Columns were centrifuged at 3,185 g for 1 min, and the flow through was discarded. To each column 700 µl of 70% (v/v) ethanol was added and incubated at room temperature for 1 min, then centrifuged at 3,185 g for 1 min, and the flow through discarded. The columns were dried by centrifugation at 3,185 g for 1 min. Columns were then placed in fresh sterile microfuge tubes, 25 µl of DEPC-treated distilled water was added to the centre of each column and incubated at room temperature for 5 min. The columns were then centrifuged at 3,185 for 1 min. Again, 10 µl of DEPC-treated distilled water was added

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to the centre of each column and incubated at room temperature for 5 min. The columns were then centrifuged at 3,185 g for 1 min. The collected volumes of approx 35 µl of the labelled cDNA were reduced to 10 µl using a speedivac.

Finally, the two labelled cDNA preparations were combined in one tube to give total volume of 20 µl. These solutions were mixed thoroughly by repetitive pipetting. Each was then reduced from 20 µl to 5 µl using a speedivac at low speed for approximately 10 – 15 min. Finally, 85 µl of hybridisation solution was added to each tube (from Table 3.4) and incubated at 65°C for 5 min. These solutions were then cooled at room temperature in darkness. Samples of ~90 µl of the labelled cDNAs were loaded onto each slide in darkness and the slides were incubated in darkness at 37°C for 16 hrs.

Table 3.4: Hydridization solution mix per slide

Reagents Volume (µL)

Dig Easy Hyb (filtered) 100

**Yeast tRNA (10 mg/ml) 5

*Salmon Herring Sperm (10 mg/ml) 2.75

Total vol. 107.75

Suppliers: *Invitrogen, **Ambion

3.3.3.4 Washing and blocking microarray slides

Microarray slides were placed in a slide chamber which was then filled up with solution 1 (0.1% Tritron X-100), and agitated gently for 5 min. Solution 1 was removed, then solution 2 (4.38 mM HCl) was added and agitated gently for 2 min. Solution 2 was removed and fresh solution 2 was added and agitated gently for 2 min. Solution 2 was removed and solution 3 (100 mM KCl) was added and agitated gently for 10 min. Solution 3 was removed and DEPC-treated distilled water was added and agitated gently for 1 min. DEPC-treated distilled water was removed and blocking buffer (25% Ethyene glycol and 0.01% HCl) was added and agitated gently at 50°C for 30 min. The blocking buffer was removed and DEPC-treated distilled water was added and agitated gently for 1 min. Slides were then air dried by centrifugation in falcon tubes at 830 g

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for 10 min at 40°C using a swing out centrifuge rotor (Sorvall® _{RT 7). Dry slides were} placed at room temperature in a light blocking slide box until required. Formula for all solutions used for washing are provided in Appendix I, section 1.1.

3.3.3.5 Hybridized slide washes

Following overnight incubation of the slides with the labeled cDNA, the cover-slip was removed by immersing in 1 x SSC + 0.1% SDS in a 50 ml falcon tube; the cover-slip was allowed to float off. The slide/s were then placed into 50 ml of 1 X SSC + SDS in a falcon tube, and this was gently agitated for 20 min. The wash solution was removed and this step repeated twice. Following the above three washes, further washes were performed in following solutions:

1 X SSC + SDS with gentle agitation for 15 min. 1 X SSC with gentle agitation for 10 min.

1 X SSC + Triton X-100 with gentle agitation for 10 min. 1 X SSC with gentle agitation for 10 min.

0.5 X SSC with gentle agitation for 10 min. This last step was repeated.

Washed slides were air dried by centrifuging at 830 g (using Sorvall® RT 7 Centrifuge) for 10 min at 40°C. Labelled slides were stored in the dark. Prior to scanning, the back of each slide was cleaned with 95% ethanol to remove any dust or smudges. Slides were scanned using a GenePix-Pro 4000, scanning machine (Axon), at the Australian Genomic Research Facility (AGRF).

3.3.3.6 Analysis of micorarray

Unreliable signals were filtered out and data normalized using GenePix Pro 5.0 software. GeneSpring software was used to determine genes as more- or less-highly expressed when the difference in expression level between stressed and control cultures was reproducibly greater than three-fold in at least two replicates. Furthermore, to minimize systematic variations within or between slides (e.g. dye incorporation differences) in the measured gene expression levels of the two co-hybridized mRNA samples, the Locally Weighted Scatterplot Smoothing (LOWESS) normalization method was used. The Saccharomyces cerevisiae genome database (SGD;

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http://www.yeastgenome.org) and yMGV (http://transcriptome.ens.fr/ymgv/) were used to group ORFs according their molecular and biological function.