|
| Status |
Public on Nov 05, 2010 |
| Title |
LUNG_3_0 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LUNG_3_H
|
| Organism |
Sus scrofa |
| Characteristics |
tissue: lung
sample type: high (H) PRRSV buden pigs
|
| Treatment protocol |
Hampshire by Duroc (HD) cross pigs were used in this study (Petry et al., 2005, 2007). Animals were inoculated intranasally (1 cc per nostril) with 105 cell culture infectious dose 50% (CCID50) of PRRSV strain 97-7985 (Osorio et al., 2002). Lung and bronchial lymph node (BLN) tissues were collected at necropsy (day 14) and stored in –80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen 3-mm3 tissue sections were rapidly homogenized in TRIzol® (Invitrogen) using a mortar and pestle. RNA was extracted from homogenized samples (PureLinkTM kit, Invitrogen). All RNA samples were DNAse-treated, evaluated for RNA quantity using Nanodrop Spectrophotometer 2000c (Peqlab) and for RNA quality using Bioanalyzer (Agilent) which results in an RNA Integrity Number (RIN) higher than 8.5.
|
| Label |
Cy3
|
| Label protocol |
For each sample, 1 µg of total RNA was subjected to the first strand cDNA synthesis with a T7 oligo(dT) primer using Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion Inc.) according to the manufacturer’s instructions. For second-strand synthesis, the cDNAs were in vitro transcribed to synthesize multiple copies of amino allyl-modified aRNAs (aRNAs). After purification 10 µg of aRNAs were labeled with Alexa Fluor®555 and Alexa Fluor®647 dyes (Invitrogen). The measurement of the final dye incorporation in labeled, purified samples was carried out using NanoDrop.
|
| |
|
| Channel 2 |
| Source name |
LUNG_0_H
|
| Organism |
Sus scrofa |
| Characteristics |
tissue: lung
sample type: pooled reference
|
| Treatment protocol |
Hampshire by Duroc (HD) cross pigs were used in this study (Petry et al., 2005, 2007). Animals were inoculated intranasally (1 cc per nostril) with 105 cell culture infectious dose 50% (CCID50) of PRRSV strain 97-7985 (Osorio et al., 2002). Lung and bronchial lymph node (BLN) tissues were collected at necropsy (day 14) and stored in –80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen 3-mm3 tissue sections were rapidly homogenized in TRIzol® (Invitrogen) using a mortar and pestle. RNA was extracted from homogenized samples (PureLinkTM kit, Invitrogen). All RNA samples were DNAse-treated, evaluated for RNA quantity using Nanodrop Spectrophotometer 2000c (Peqlab) and for RNA quality using Bioanalyzer (Agilent) which results in an RNA Integrity Number (RIN) higher than 8.5.
|
| Label |
Cy5
|
| Label protocol |
For each sample, 1 µg of total RNA was subjected to the first strand cDNA synthesis with a T7 oligo(dT) primer using Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion Inc.) according to the manufacturer’s instructions. For second-strand synthesis, the cDNAs were in vitro transcribed to synthesize multiple copies of amino allyl-modified aRNAs (aRNAs). After purification 10 µg of aRNAs were labeled with Alexa Fluor®555 and Alexa Fluor®647 dyes (Invitrogen). The measurement of the final dye incorporation in labeled, purified samples was carried out using NanoDrop.
|
| |
|
| |
| Hybridization protocol |
Labeled aRNAs were purified, fragmented and denatured at 95°C for 10 min., combined according to the experimental design, and mixed with 50 µl of SlideHyb Buffer (SlideHybTM Glass Array Hybridization Buffer #1, Ambion Inc.). Pigoligoarray hybridizations were performed in sealed hybridization cassettes (ArrayIt, TeleChem International, Inc.) for 18 h at a humid 43°C . Following hybridization, microarray slides were washed with low (2xSSC and 0.5% SDS) and high (0.2xSSC and 0.2% SDS) stringency wash buffers for 10 minutes each.
|
| Scan protocol |
Fluorescent images were detected by an Axon GenePix® 4000B scanner (Molecular Devices, Sunnyvale, CA, USA) and fluorescence intensity data were collected using GenePix® Pro 6 software (Molecular Devices) after spot alignment.
|
| Data processing |
Median intensities were extracted and normalized using a within print-tip lowess location normalization and an overall scale normalization. The resulting normalized data were expressed in the log2 scale.
|
| |
|
| Submission date |
Nov 03, 2010 |
| Last update date |
Nov 04, 2010 |
| Contact name |
juan p steibel |
| E-mail(s) |
[email protected]
|
| Organization name |
Michigan State University
|
| Street address |
1205 I Anthony Hall
|
| City |
East Lansing |
| State/province |
MI |
| ZIP/Postal code |
48842 |
| Country |
USA |
| |
|
| Platform ID |
GPL7435 |
| Series (1) |
| GSE25120 |
Identifying putative candidate genes and pathways involved in immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection |
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