Microarray analysis

The setup of the microarray experiment was as depicted in figure 4.2 with Ge-tra and Ge-tet-1 both untreated and treated with doxycycline for 24 hrs. Technical replicates and dye-swaps were made to ensure high reliability of the results.

Figure 4.2: Setup of the microarrayThe control clone Ge-tra was compared with the doxycycline treated Ge-tra and the inducible clone Ge-tet-1 with the clone Ge-tet-1 acutely overexpressing HSP70 after doxycycline treatment for 24 hrs. Both comparisons were made in technical replicates and with dye swaps, meaning that in 2 out of 4 arrays the untreated sample was labelled with Cy3 and in the other 2 the treated samples. In total 8 whole human genome microarrays with 44000 60-mer oligos were used.

Isolated RNAs from Ge-tra and Ge-tet-1 cells from three different treatments with doxycycline were pooled in equal amounts (500 ng each). As the RNA integrity was already determined, the first step of the oligo two-colour microarray was to amplify and to label the target RNA to generate complementary cRNA. This was achieved in two sequential reactions. In the first reaction, unlabelled dsDNA is generated from mRNA primed with an oligo (d)T-T7 promoter primer by reverse transcription using

MMLV-4 Methods

reverse transcriptase without amplification. In the second reaction, Cy3 or Cy5-labelled amplified single-stranded cRNA is generated by a T7 RNA polymerase using an anti-sense promoter as depicted in 4.3 on the following page.

For the preparation of the transcription of mRNA into dsDNA 2µl of each 1:3200 diluted Spike-mix A or B were denatured together with 8.3 µl RNA (1500 ng) and 1.2 µl of T7 promoter primer for 10 min at 65^◦C in a thermocycler. The reactions were placed on ice afterwards for 5 min, while the cDNA master mix was prepared (table 4.1). The addition of Spike-mix A and B serves the purpose of an internal quality control. They contain 10 different sequences of AdV in different concentrations, which bind to distinct dots on the microarray. On the basis of their fluorescence intensities and ratios between each other, it can be deduced whether the labelling and the hybridisation worked optimally for all samples.

Table 4.1: Master mix for dsDNA for microarrayTo generate dsDNA from mRNA.

Master mix for the generation of dsDNA per reaction 4 µl 5 ×First strand buffer

2 µl 0.1 M DTT 1 µl 10 mM dNTP mix

1 µl MMLV-reverse transcriptase 0.5µl RNaseOUT

The denatured reactions and the cDNA master mix were mixed and all samples were incubated in a thermocycler. First the samples were incubated for 2 hrs at 40^◦C and then for 15 min at 65^◦C, before placing them for 5 min on ice. The generation of dsDNA was followed by the generation of amplified labelled cRNA by T7 RNA polymerase. To achieve that, each dsDNA sample was mixed with 60 µl of transcription master mix (table 4.2) and incubated for 2 hrs at 40^◦C in a thermocycler.

Table 4.2: Master mix for generating labelled cRNA for microarray To be able to distinguish the two different cRNAs, added to the array later on, they need to be labelled with diferrent dyes, here Cyanine 3-CTP or Cyanine 5-CTP.

Master mix per 60 µl reaction to generate labelled cRNA 20 µl 4×Transcription buffer

2.4µl Cyanine 3-CTP or Cyanine 5-CTP 0.5µl RNaseOUT

Ad 60µl RNase-free H₂O

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Figure 4.3: Generation of labelled cRNA for a two-colour microarrayThe scheme shows the amplification of mRNA into double-stranded cDNA and from this the generation of either Cy3 or Cy5-labelled cRNA. The scheme is taken from the Agilent technologies protocol “Two-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling)” version 5.7 from March 2008.

4 Methods

After producing amplified labelled cRNA, the next step was to purify and to quantify it, in order to apply the different cRNA samples in the right amount to the array. For the purification of the amplified RNA Qiagen’s RNeasy Mini kit was used. Briefly, the volume of each sample was filled to a final volume of 100µl with RNase-free water, 350µl RLT buffer were added and well mixed. 250µl 100% EtOH were added to the tubes and mixed by pipetting up and down to precipitate the RNA. Samples were transferred to RNeasy mini columns, centrifuged for 30 sec at 4 ^◦C at 12300 x g and the through-flow was discarded. The columns were transferred into new collection tubes, and after washing the columns twice with 500µl EtOH-containing RPE buffer for 30 sec and 1 min at 4^◦C at 12300 x g, the columns were transferred into new 1.5 ml tubes. Finally, RNA was eluted by adding 30µl RNase-free water directly onto the column, waiting for 1 min, followed by centrifugation for 30 sec at 4^◦C at 12300 x g.

This purified RNA was then quantified using NanoDrop ND-1000. The exact yield of labelled RNA per sample was calculated with the measured concentration of cRNA using formula 4.6, whereby the elution volume was 30 µl.

µg cRNA yield = (concentration of cRNA)×(elution volume)

1000 (4.6)

With the measured values for the concentration of Cy3 and Cy5 and formula 4.7 the specific activity defined as pmol dye perµg cRNA can be calculated.

specific activity [pmole/µg] = concentration of Cy3 or Cy5 [pmole/µl]

concentration of cRNA [ng/µl]×1000 (4.7) A yield of less than 825 ng cRNA per array and a specific activity of less than 8.0 pmole/µg is not sufficient and the cRNA preparation needs to be repeated in order to proceed. This was not the case for any of our samples (see table B.1 on page 171).

The next step was to prepare the 10×blocking agent and the hybridisation samples for the microarray. The blocking agent was solubilised by adding 500µl RNase-free H2O to a tube containing lyophilised 10×blocking agent supplied with the Agilent Gene Expression Hybridization Kit and by vortexing gently. The sizes of the cRNA hybridisation samples for the microarray ranged between 50 and 3000 bases and were fragmented into shorter ones with an optimal size of 50 to 200 bases to improve target specificity to the 60mer oligos of the microarray. Hybridisation samples were fragmented by exposure to zinc acetate, which is the main component of the fragmentation mix given in table 4.3 on the following page. cRNA was mixed with the fragmentation mix in a 1.5 ml tube and placed in the pre-warmed hybridisation oven for exactly 30 min at 60^◦C. The reaction was quenched by carefully mixing the fragmented cRNA with 55 µl 2× GE hybridisation buffer Hi-RPM, which contains an excess of EDTA.

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Table 4.3: Fragmentation mix for one 4×44K microarrayFor the fragmentation of the labelled cRNAs, the following components needed to be mixed for each one of the four arrays. Either Cy3 or Cy5-labelled cRNA was used in the different fragmentation mixes.

55 µl fragmentation mix for one 4×44K microarray 825 ng Cy3-labelled cRNA

825 ng Cy5-labelled cRNA 11 µl 10×blocking agent 2.2µl 25×fragmentation buffer ad 50 µl RNase-free H₂O

The hybridisation assembly was performed in an ozone-free room and nitrile gloves were worn during working with the arrays. Clean gasket slides (backing) were loaded into Agilent SureHyb chamber bases with the labels facing up. The hybridisation samples, 100 µl each, were slowly dispensed onto the backing without touching its walls. Two arrays with the “active side” down were carefully placed onto the backings, so that the numeric barcodes were facing up. Afterwards, the SureHyb chamber covers were placed onto the sandwiched slides and the clamps were handtighten onto the chambers. The assembled chambers were rotated vertically to wet the backing and to assure the mobility of air bubbles. The slide chambers were placed for 17 hrs into the hybridisation oven set to 65 ^◦C at 10 rounds per minute (rpm).

After 17 hrs of hybridisation, the slides were washed with two different wash solutions.

1000 ml wash solution 1 containing 6×SSPE, 0.005 % N-lauroylsarcosine in H₂O were used for 8 arrays and 500 ml of wash solution 2 with 0.06×SSPE, 0.005 % N-lauroylsarcosine in H₂O were used. A series of washing steps was prepared: firstly 250 ml wash solution 1, secondly 250 ml wash solution 1 on a stirrer, thirdly 250 ml wash solution 2 on a stir-rer, followed by 250 ml acetonitrile. Hybridisation chambers were dissembled in washing solution 1 and ”active sites” were collected in a slide rack in wash solution 1 on a stirrer.

The analysis of the microarray was done by scanning the arrays with a resolution of 5µm and two different laser intensities with a scanner from Agilent Techonologies. First, the laser scanned the arrays with 100 % intensity and then again with 10 % intensity to get reliable data of all spots, which were overamplified with 100 % laser intensity. The data of the scanned arrays were then exported with the software accompanying the scanner and distinct algorithms were used in R to normalise the data and to calculate the logarithmic expression data and p-values.