cell line: capillary endothelial cells time: 3h (nontransformed cell line) agent: control tissue: Adrenal Medulla genotype/variation: Wild Type
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated as TRIzol method. RNA concentration and integrity were detected using the Agilent's 2100 Bioanalyzer with an RNA 6000 Nano LabChip® Kit. Confirmatory readings were performed using the ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE).
Label
Cy3
Label protocol
Total RNA from control or experimental samples was linearly amplified and labeled using two different fluorescent dyes: cyanine 3-CTP (Cy-3), and cyanine 5-CTP (Cy-5, Amersham Biosciences) as described in the Agilent Low RNA Input Linear Amplification Kit. Briefly, in a first step, double stranded cDNA (dscDNA) was generated using the Moloney Murine Leukaemia virus reverse transcriptase (MMLV-RT). In the first reaction, 1 μg of total RNA was mixed with T7 promoter primers and the diluted Spike-in Mix (A or B), incubated at 65˚C during 10 min and placed on ice for 5 min. The denatured primers and total RNA were mixed with the following reagents: 5 X First Strand Buffer, 0.1 M DTT, 10 mM dNTP mix, MMLV-RT, and RNaseOUT. This reaction mix was incubated at 40˚C for 2 hrs, then at 65˚C for 15 min, and finally placed on ice for 5 min. Next, synthesis and labeling of cRNA was performed simultaneously mixing the prepared reaction (containing the dscDNA) with a transcription Master Mix: (4 X Transcription Buffer, 0.1 M DTT, NTP mix, 50% PEG, RNAseOUT, inorganic pyrophosphatase, T7 RNA polymerase, and Cy-3 or Cy-5 (10 mM, each). The reaction was incubated at 40˚C for 2 hrs. Amplified fluorescent cRNA probes were purified using the Qiagen's RNeasy-mini spin columns. Finally, fluorescently labeled cRNA samples were analyzed for yield and dye incorporation using the ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE). Only samples that presented a concentration of more than 8 pmol/mg (a measure representing efficient dye incorporation) were used for hybridization as recommended by the manufacturer.
Total RNA was isolated as TRIzol method. RNA concentration and integrity were detected using the Agilent's 2100 Bioanalyzer with an RNA 6000 Nano LabChip® Kit. Confirmatory readings were performed using the ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE).
Label
Cy5
Label protocol
Total RNA from control or experimental samples was linearly amplified and labeled using two different fluorescent dyes: cyanine 3-CTP (Cy-3), and cyanine 5-CTP (Cy-5, Amersham Biosciences) as described in the Agilent Low RNA Input Linear Amplification Kit. Briefly, in a first step, double stranded cDNA (dscDNA) was generated using the Moloney Murine Leukaemia virus reverse transcriptase (MMLV-RT). In the first reaction, 1 μg of total RNA was mixed with T7 promoter primers and the diluted Spike-in Mix (A or B), incubated at 65˚C during 10 min and placed on ice for 5 min. The denatured primers and total RNA were mixed with the following reagents: 5 X First Strand Buffer, 0.1 M DTT, 10 mM dNTP mix, MMLV-RT, and RNaseOUT. This reaction mix was incubated at 40˚C for 2 hrs, then at 65˚C for 15 min, and finally placed on ice for 5 min. Next, synthesis and labeling of cRNA was performed simultaneously mixing the prepared reaction (containing the dscDNA) with a transcription Master Mix: (4 X Transcription Buffer, 0.1 M DTT, NTP mix, 50% PEG, RNAseOUT, inorganic pyrophosphatase, T7 RNA polymerase, and Cy-3 or Cy-5 (10 mM, each). The reaction was incubated at 40˚C for 2 hrs. Amplified fluorescent cRNA probes were purified using the Qiagen's RNeasy-mini spin columns. Finally, fluorescently labeled cRNA samples were analyzed for yield and dye incorporation using the ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE). Only samples that presented a concentration of more than 8 pmol/mg (a measure representing efficient dye incorporation) were used for hybridization as recommended by the manufacturer.
Hybridization protocol
The hybridization and washing protocol were done as recommended by the manufacturer (Two-Color Microarray-Based Gene Expression Analysis Protocol, Version 5.5, February 2007, from Agilent Technologies). Briefly, 300 ng of Cy-3 and Cy5-labeled cRNAs were combined with 10 X Blocking Agent, and 25 X Fragmentation Buffer at 60˚C for 30 min. The reaction was stopped with 2 X GEx Hybridization Buffer HI-RPM and hybridized to the oligo microarray for 17 hrs at 60˚C with continuing rotation (10 rpm). The reagents used for this step were from the Gene Expression Hybridization Kit. After hybridization, slides were washed during 1 min at RT using the GE Wash Solution 1, and 1 min at 37˚C with GE Wash Solution 2. Next, slides were washed for 1 min with acetonitrile (Sigma). A final washing was done using a Stabilization and Drying Solution for 30 sec.
Scan protocol
Data images were obtained by laser-based scanning using the Microarray Scanner and the Feature Extraction Software (v8.1) both from Agilent. Scanning parameters were as recommended by the manufacturer. Briefly, the resolution settings for scanning were 5 micrometers using minimum (10%) and maximum (100%) Photo Multiplier Tube detection sensitivities. An initial assessment of the array was performed using the information from the Quality Report extracted from the Feature Extraction Software (v8.1).
The analysis of the microarray data consisted of the following steps: 1) within-array and 2) between-array normalization. Normalization of the data was as previously described (Smyth and Speed 2003). Briefly, in the “within-array normalization” analyses the M-values were normalized for each array separately using a global loess method without background correction, whereas in the “between-array normalization” analyses log-ratios were normalized to be comparable across arrays using the Aquantile method. Normalization success was determined using MA plot graphical representations. These MA-plots depict the log-ratio of Red versus Green (the M value in the y axis) against the overall intensity of each spot (the A value in the x axis). The log-ratio is represented by the M-value, M = log2(R) _ log2(G), and the overall intensity by the A-value, A = (log2(R) +log2(G))/2. In this experiment, RNA sources from different time points after Tunicamycin exposure were included (at 3 hrs and 32 hrs). Dye swapping was done to correct for differences in the incorporation of the dyes. The main comparisons were done for each time point against the control sample and the Tunicamycin treated sample.
