22A scrapie infected (intracranial; IC) VM mouse whole brain at end point (>200 dpi).
Extracted molecule
other
Extraction protocol
Low molecular weight (LMW) RNA enrichment from whole mouse brain was performed using mirVana™ miRNA Isolation Kit (Ambion), according to the manufacturer's protocol. LMW RNA was DNase-treated for microarray target preparation using TURBO DNA-free™ (Ambion).
Label
Cy3
Label protocol
Array 900 miRNA RT Kit (Genisphere) was used to prepare labelled cDNA targets for microarray hybridization according to the manufacturer's protocol. Briefly, 250 ng of LMW enriched RNA was used as a template in a 100 μl Poly(A) Tailing and RT reaction containing 1× Reaction Mix, 2.5 mM MnCl2, 1 mM ATP, and 4 μL poly A polymerase (E-PAP Enzyme). After incubation at 37°C for 15 minutes the reaction was placed on ice and 2 μL of Cy3 or Cy5 reverse transcriptase primer was added. The reaction mix was incubated at 65°C for 10 minutes prior to addition of 23 μL of a second master mix containing per reaction: 10 μL of 5× First Strand Buffer, 5 μL of 0.1 M DTT, 2.5 μL of dNTP Mix, 1 μL of Superase-in RNase Inhibitor, 2 μL of SuperScript II Reverse Transcriptase (200 U) (Invitrogen), and 2.5 μL of nuclease-free water. Subsequently, the reaction was incubated at 42°C for one hour. Finally, 8.75 μL of 0.5 M NaOH/50 mM EDTA and 65°C for 15 minutes was used to inactivate Superscript II. Samples were concentrated to a volume of approximately 10–15 μL, using Microcon YM-10 Centrifugal Filter Devices (Fisher Scientific) according to the manufacturer's protocol.
Channel 2
Source name
PBS treated (IC) VM mouse (age matched) whole brain.
PBS treated (intracranial; IC) VM mouse (age matched) whole brain at end point (>200 dpi).
Extracted molecule
other
Extraction protocol
Low molecular weight (LMW) RNA enrichment from whole mouse brain was performed using mirVana™ miRNA Isolation Kit (Ambion), according to the manufacturer's protocol. LMW RNA was DNase-treated for microarray target preparation using TURBO DNA-free™ (Ambion).
Label
Cy5
Label protocol
Array 900 miRNA RT Kit (Genisphere) was used to prepare labelled cDNA targets for microarray hybridization according to the manufacturer's protocol. Briefly, 250 ng of LMW enriched RNA was used as a template in a 100 μl Poly(A) Tailing and RT reaction containing 1× Reaction Mix, 2.5 mM MnCl2, 1 mM ATP, and 4 μL poly A polymerase (E-PAP Enzyme). After incubation at 37°C for 15 minutes the reaction was placed on ice and 2 μL of Cy3 or Cy5 reverse transcriptase primer was added. The reaction mix was incubated at 65°C for 10 minutes prior to addition of 23 μL of a second master mix containing per reaction: 10 μL of 5× First Strand Buffer, 5 μL of 0.1 M DTT, 2.5 μL of dNTP Mix, 1 μL of Superase-in RNase Inhibitor, 2 μL of SuperScript II Reverse Transcriptase (200 U) (Invitrogen), and 2.5 μL of nuclease-free water. Subsequently, the reaction was incubated at 42°C for one hour. Finally, 8.75 μL of 0.5 M NaOH/50 mM EDTA and 65°C for 15 minutes was used to inactivate Superscript II. Samples were concentrated to a volume of approximately 10–15 μL, using Microcon YM-10 Centrifugal Filter Devices (Fisher Scientific) according to the manufacturer's protocol.
Hybridization protocol
Tagged-cDNA hybridization followed the protocol outlined in the 900 miRNA RT Kit. A hybridization mixture consisting of the differentially tagged cDNA (10 μL of Cy3-labelled and 10 μL of Cy5-labelled targets) and 2 × SDS-based Hybridization Buffer pre-heated to 70°C (20 μL) was mixed and incubated at 75–80°C for 10 minutes, cooled to 50°C until loading and added to the microarray; specifically a 22 × 40 mm cover slip (mSeries Lifterslip™) (Erie Scientific) was centered over the grids and the preheated hybridization mixture was loaded under the cover slip. Microarrays were incubated overnight (16–20 hours) at 50°C in a dark humidified chamber (Genetix). Following hybridisation the cover-slips were removed and the arrays were washed in 2 × SSC, 0.2% SDS wash buffer preheated to 42°C for 15 minutes, 2 × SSC wash buffer at room temperature for 10–15 minutes, and 0.2 × SSC wash buffer at room temperature for 10–15 minutes. Arrays were dried by centrifugation at 1000 rpm for 2–3 minutes and the 3DNA system containing the fluorescent cyanine molecules were hybridized to the arrays; in this case the hybridization mixture contained Cy3 3DNA Capture Reagent (2.5 μL), Cy5 3DNA Capture Reagent (2.5 μL), Nuclease Free Water (15 μL), and 2 × SDS-based Hybridization Buffer. The mix was heated to 70°C for 10 minutes, cooled to 62–64°C and hybridized to the arrays for 4 hours at 62–64°C in a dark humidified chamber. Finally, the arrays were washed as previously described.
Scan protocol
Microarrays were scanned using Agilent G2565AA and Agilent G2565BA Microarray Scanner System (Agilent). Feature extraction was performed using Array-Pro™ analyzer version 4.5 (Media Cybernetics).
Description
no additional information
Data processing
Microarrays were scanned using Agilent G2565AA and Agilent G2565BA Microarray Scanner System (Agilent). Feature extraction was performed using Array-Pro™ analyzer version 4.5 (Media Cybernetics). Data were stored and normalized in the GeneTraffic Microarray Database and Analysis System (Iobion Informatics, La Jolla, CA). The raw data was filtered so that individual spots had to pass a number of quality criteria including, minimum intensity levels, minimum signal to background ratios, and no greater than 10% missing values for each gene. Genes who passed these criteria were used for further data analysis. Each slide was then normalized using a linear regression, smoothing algorithm (Loess best-fit) over individual array sub grids. Log2 ratios were used for further statistical analysis.
