3. METHODS
3.5. DNA Microarrays
Methods
amplified again with the primers 5’_Luc_BA5_HindIII and 3’_Luc_BA5_SpeI, restricted with HindIII and SpeI and ligated into the same sites of the pMIR-Report vector. Clones were analyzed and two clones containing an insert of appropriate size for one, as well as four binding sites were sequenced.
3.5.1. cDNA Synthesis with Spike-in Controls
Control RNA and sample RNA including spike in controls (one for each dye: spike A for Cy3, spike B for Cy5) were transcribed into cDNA. Spike in RNAs were first diluted according to the manufacturer’s instructions 1:20 and afterwards to 1:40. From the last dilution 2 μl were mixed with 1.2 μl T7 promotor primer and 1 μg sample RNA in 8.3 μl DEPC-H2O, heated to 65 °C for 10 min and incubated on ice for 5 min. Subsequently 8.5 μl of a cDNA mix were added.
Table 3-50 cDNA Synthesis for Expression Arrays
Reagents Amount
5x FS Buffer 4 μl
DTT [0.1 M] 2 μl
dNTPs [10 mM] 1 μl
MMLV-RT 1 μl
RNAseOut 0.5 μl
The cDNA synthesis was performed at 40 °C for 2 h. Afterwards the reverse transcriptase was inactivated by heating the samples to 65 °C for 15 min and incubated on ice for 5 min.
3.5.2. cRNA Synthesis with Fluorescence Dye Incorporation
After cDNA synthesis, 60 μl of transcription master mix (listed in table 3-51) were added.
Table 3-51 cRNA Synthesis and Labeling of RNA for Expresion Arrays
Reagents Amount
DEPC-H20 15.3 μl
4x Transcription buffer 20 μl
DTT [0.1 M] 6 μl
NTP-Mix 8 μl
50% PEG 6.4 μl
Anorganic Phosphatase 0.5 μl T7-RNA Polymerase 0.6 μl
RNAseOut 0.8 μl
Cyanine 3-CTP or Cyanine 5-CTP 2.4 μl
The cRNA synthesis was carried out at 40 °C for 2 h. Samples were purified with the RNeasy Kit and purity and dye incorporation were measured spectrophotometrically. The total amount of RNA was calculated with the following formula.
cRNA of volume g
elution l
cRNA of ion
Concentrat ×
μ
=μ
1000
) (
30 ) (
The specific activity was calculated with the following formula:
cRNA g per Cy or Cy Cy pmol
or Cy of ion
Concentrat 3 5) 1000 3 5
μ
( × =
Methods
Only if the reaction yielded more than 825 ng cRNA and if the specific activity was greater than 8 pmole per μg cRNA, the samples were hybridized to the array. Otherwise the labeling reaction was repeated.
3.5.3. Hybridization of DNA Microarrays
The same amount of labeled Cy3 and Cy5 cRNA were mixed with the reagents listed in table 3-52.
Table 3-52 Hybridization Mixture for Expression Arrays
Reagents Amount
Cy-3 labeled cRNA 825 ng Cy-5 labeled cRNA 825 ng 10x Blocking solution 11 μl
DEPC-H2O ad 52.8 μl
25x Fragmentation buffer 2.2 μl
The mixture was incubated for 30 min at 60 °C, immediately mixed with one volume of 2x GEx Hybridization Buffer HI-RPM and loaded onto the array. Incubation of the array was performed for 17 h at 65 °C and 10 rpm in a hybridization oven slowly rotating.
3.5.4. Washing of DNA Microarrays
After disassembling the chamber and dissociating the gasket slide, the array was washed in a glass container filled with wash buffer 1. The array was transferred carefully with tweezers in a second glass container with a holder for the array filled with wash buffer 1. A magnetic stir bar under the holder was slowly rotating for 1 min. Afterwards the holder containing the array was transferred into a third glass container with magnetic stir bar and prewarmed wash buffer 2 and incubated for 1 min under stirring. Then the holder was taken out carefully and very slowly (10 s) allowing the array to dry and then was shortly centrifuged on a cell culture dish holder (300 rpm, 2 min, RT, Heraeus). The array was immediately scanned on a GenePix reader 4100A.
3.5.5. Scanning and Evaluation of DNA Microarrays 3.5.5.1. Scanning
The GenePix 4100A uses a high-sensitivity, low-noise photo multiplier tube (PMT) to detect the emitted light.
A preview scan with 40 μm resolution was performed to automatically determine the PMT gain for each wavelength. After that the first scan was performed using a 5 μm spatial resolution with the calculated optimal PMTs and then 2-3 further scans were done using higher and lower PMTs. The
dynamic range of a detection is the range of signal values over which the instrument can accurately measure changes and is often considered in conjunction with its detection limit. The detection limit is defined as the dye concentration for which the signal to noise ratio is 3. The signal to noise ratio is calculated with the following formula:
Background of
ion darddeviat S
Background Signal
Ratio Noise to Signal
tan
)
( −
=
3.5.5.2. Evaluation
To get more reliable results, two different analysis softwares, the GenePix software and the Feature Extraction software in combination with GeneSpring GX10 were used.
For GenePix analysis, the image and grid file were opened simultaniously in the program. After placing the grid exactly over the spots, features in a selected block were aligned and analyzed.
Integrated settings like “fair features” were further used to flag spots as “good” when fulfilling the fair feature criteria. All “good” flagged spots were included for normalization and control spots were excluded. Normalization was performed using the ratio of medians of all normalization features to yield an overall ratio of 1. Results were exported as .txt or .gpr file for further analysis. The cut off was calculated using the genome browser. Multiple spots of the same substance were averaged. This was performed with Microsoft Excel for either one or two arrays. For biological duplicate experiments a standard deviation was calculated for each substance.
Feature Extraction (FE) consists of two major processes, first the image analysis to place the grid and locate spots and second the data analysis to define and measure spot features for gene expression. FE automatically detects the array barcode and loads the corresponding grid template and an extraction protocol. The grid template was automatically fitted to the spots and the FE extraction protocol was applied to carry out data analysis.
During data analysis FE removes outlier pixels and calculates statistics of inlier pixels of features and background. The software subtracts the background from the features. It also estimates the error at this point. With the 2-color experiments, it performs dye normalization and for gene expression calculates a p-value, that is a confidence measure of differential gene expression.
Gene Spring GX10 was afterwards used to analyze the FE data from the Agilent expression arrays.
Arrays were treated as duplicates or triplicates by giving them just one parameter in the experiment grouping step (e.g. Adeno infected = yes). Probe sets were then filtered by their flag values (analyzed by Feature Extraction) P(present), M(marginal) and A(absent). Only entities having the present and marginal flags in at least two or all samples are displayed in a profile plot. The significance analysis was performed afterwards using the T-test against 0 without correction. Fold change analysis was finally used to identify genes with expression ratios or differences between miRNA expressing cells and control cells, that are outside of a given cut off or threshold. The cut off was arbitrarily set to 1.2
Methods