Agilent microarray analysis

Transcriptome analysis was performed using Agilent 8×60 K microarrays followed the

workflow of sample preparation and array processing (Figure 34).

109 Figure 34. Scheme of sample preparation and array processing

4.2.2.5.1 One-color Spike Mix preparation

One-Color Spike Mix stock solution was vigorously mixed by a vortex mixer and was heated at 37°C for 5 min. The One-Color Spike Mix stock solution was vigorously mixed once again using a vortex mixer and was then briefly centrifuged. The first dilution was generated by mixing 2 µl of One-Color Spike Mix stock solution with 38 µl of Dilution Buffer, mixed thoroughly using a vortex mixer and then briefly centrifuged. The second dilution was generated by mixing 2 µl of the first dilution with 48 µl of Dilution Buffer, mixed thoroughly using a vortex mixer and again briefly centrifuged. The third dilution was created by mixing 2 µl of the second dilution with 38 µl of Dilution Buffer, mixed thoroughly using a vortex mixer and again briefly centrifuged.

Total RNA

cDNA synthesis

cRNA synthesis and amplification

cRNA purification

Hybridization

Wash

Scan

Feature extraction

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4.2.2.5.2 Labeling reaction preparation

Two microliter of the third dilution of One-Color Spike Mix was pipetted into 100 ng of each total RNA sample. Then 1.8 µl of T7 Promoter Primer Mix containing 0.8 µl of T7 Promoter Primer and 1 µl of Nuclease-free water were pipetted into the mix of spike dilution and total RNA. The mix was incubated in a circulating water bath at 65°C for 10 min to denature the primers as well as the templates and then was incubated on ice for 5 min. Afterwards, the 5 × first strand buffer was prewarmed at 80°C for 5 min until the buffer components were resuspended. Then 4.7 µl of cDNA Master Mix containing 2 µl of 5 × first strand buffer, 1 µl of 0.1 M DTT, 10 mM dNTP mix and 1.2 µl of AffinityScript RNase Block Mix were added into each sample tube and were gently mixed by pipetting. The mix was incubated in a circulating water bath at 40°C for 2 h followed by incubation at 70°C for 15 min and was then placed on ice for 5 min. Finally, 6 µl of Transcription Master Mix containing 0.75 µl of Nuclease-free water, 3.2 µl of 5 × Transcription Buffer, 0.6 µl of 0.1 M DTT, 1 µl of NTP mix, 0.21 µl of T7 RNA Polymerase Blend and 0.24 µl of Cyanine 3-CTP were added into each sample tube, gently mixed by pipetting and then incubated in a circulating water bath at 40°C for 2 h (Table 20).

Table 20. Preparation of Mix

Component Volume per reaction (µl)

T7 Promoter Primer Mix T7 Promoter Primer 0.8

Nuclease-free water 1

cDNA Master Mix

5×First Strand Buffer 2

0.1 M DTT 1

10 mM dNTP mix 0.5

AffinityScript RNase Block Mix 1.2

Transcription Master Mix

Nuclease-free water 0.75

5×Transcription Buffer 3.2

0.1 M DTT 0.6

NTP mix 1

T7 RNA Polymerase Blend 0.21

Cyanine 3-CTP 0.24

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4.2.2.5.3 Labeled and amplified RNA purification

RNeasy mini spin columns (Qiagen) were used for purification of the labeled and amplified cRNA samples. Firstly, 84 µl of nuclease-free water were added into each cRNA sample. Then 350 µl of RLT Buffer and 250 µl of ethanol were added into each sample tube and mixed thoroughly by pipetting. In total 700 µl of the cRNA sample were transferred into the RNeasy mini column and were centrifuged at 13000 rpm at 4°C for 30 s. Then 500 µl of RPE buffer were as added into the RNeasy mini column after discarding the flow-through and were centrifuged at 13000 rpm at 4°C for 30 s. Another 500 µl of RPE buffer were added into the RNeasy mini column after discarding the flow-through and were centrifuged at 13000 rpm at 4°C for 60 s. To remove the remaining RPE buffer, the RNeasy mini column was transferred to a new collection tube and was centrifuged at 13000 rpm at 4°C for 30 s. Finally, 30 µl of RNase-free water were directly pipetted onto the RNeasy filter membrane and centrifuged at 13000 rpm at 4°C for 60 s after waiting for 60 s at room temperature to collect the cRNA sample.

4.2.2.5.4 cRNA quantification

The quantification of cRNA was assessed by the NanoDrop ND-1000 Spectrophotometer.

Microarray measurement was selected and measured following the procedure described in 4.2.2.4.2. The yield and the specific activity of cRNA was calculated according to the formula of (Concentration of cRNA) × 30 µl (elution volume) / 1000 = µg of cRNA and (Concentration of Cy3 / Concentration of cRNA) × 1000 = pmol Cy3 per µg cRNA, respectively. The cRNA samples with more than 0.825 µg of the yield and at least 6 pmol Cy3 per µg cRNA of the specificity activity were used for hybridization.

4.2.2.5.5 Hybridization

The 10 × Blocking Agent was incubated at 37°C for 5 min and then the Fragmentation mix

was prepared for 8-pack microarray formats by mixing 600 ng of cRNA, 5 µl of 10 × Blocking

112

Agent, Nuclease-free water to bring the volume to 24 µl and 1 µl of 25 × Fragmentation Buffer. The Fragmentation mix was incubated at 60°C in a circulating water bath for exactly 30 min and was then cooled on ice immediately for 1 min. Then the hybridization mix was prepared by adding 25 µl of 2 × GE × Hybridization Buffer HI-RPM into fragmentation mix, mixing well by pipetting, and then centrifuging at 13000 rpm at room temperature for 1 min.

Afterwards, a new gasket slide was loaded into the Agilent SureHyb chamber base with the label facing up and 40 µl of Hybridization mix were slowly dispensed in the middle of the gasket well. The array was slowly placed onto the gasket slide with the active side facing down. The cover of the SureHyb chamber was placed on the slides and hand-tightened using the clamps to assemble the chamber. The assembled chamber was vertically rotated to wet the gasket and assess the mobility of the bubbles and then incubated at 10 rpm at 65 °C for 17 h in the hybridization oven.

4.2.2.5.6 Microarray wash

The staining dishes, racks and stirs used for microarray wash should be carefully washed by

rinsing with double-distilled water (ddH

2

O) for five times. In particular, the staining dishes,

racks and stirs used for Agilent Stabilization and Drying Solution were needed to be washed

with acetonitrile for 5 min and then rinsed with ddH

2

O for five times. The staining dishes,

racks and stirs were left at room temperature for drying before use. Five staining dishes

were filled in order with Wash Buffer 1 (Dish 1), Wash Buffer 1 (Dish 2), prewarmed Wash

Buffer 2 (37°C, overnight; Dish 3), acetonitrile (Dish 4), and Stabilization and Drying Solution

(Dish 5). Then the hybridization chamber was took out of the oven and disassembled. The

array-gasket sandwich was separated by grabbing the slides from their ends in Wash Buffer 1

(Dish 1). Then the array was immediately transferred to Wash Buffer 1 (Dish 2) and placed

into the slide rack. One minute later, the slide rack was transferred to Wash Buffer 2 (Dish 3)

for 1 min followed by washing in Acetonitrile for 10 s. Then the slide rack was transferred

into Stabilization and Drying Solution (Dish 5) and was slowly moved out of the solution after

30 s.

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4.2.2.5.7 Slide scan and figure extraction

The slides were scanned using the Agilent Microarray Scanner. The slides were placed into

the slide holder with the Agilent barcode facing up. The slot number and Profile

AgilentG3_GX_1Color were set for 8 × 60 K microarray scan. The scan settings were verified

with the dye channel green, scan area (61 × 21.6 mm), scan resolution 3 µm as well as tiff

20 bit and then the scan was performed. Microarray data extraction was performed using

the Agilent Feature Extraction Software with the extract set of GE1_1010_Sep10.

Im Dokument The role of PIP aquaporins in Response to various environmental Scenarios in Arabidopsis thaliana (Seite 120-125)