|
| Status |
Public on Oct 27, 2015 |
| Title |
Lung_LBW_Rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Lung from LBW pig
|
| Organism |
Sus scrofa |
| Characteristics |
animal id: 288
birth weight category: LBW
resistance category: None
gilt id: G2216
age: 4 weeks old
|
| Treatment protocol |
Pigs were inoculated intratracheally with 1x10^7 PFU of SIV strain A/swine/Texas/4199-2/98 H3N2 (TX98) while anaesthetized, and kept in sternal recumbency for 10 minutes. Twice daily during the challenge period, rectal temperatures were taken and the animals were scored for clinical signs of the respiratory disease caused by influenza. Pigs were euthanized 48 hours post-inoculation by pentobarbital injection. Lungs and the attached trachea were removed from the animal and the percentage of lung area affected by consolidation was evaluated and used to select a subset of 'resistant' (RES) and 'susceptible' (SUS) pigs for a separate analysis. Tissue samples from each lobe were then snap-frozen in liquid nitrogen and stored at -80°C for gene expression studies.
|
| Growth protocol |
Litters for the study were selected at birth from a farm in Saskatchewan, Canada. Litter eligibility was based on the average weight of all live and stillborn piglets in the litter. High birth weight (HBW) and low birth weight (LBW) litters from parity 1-2 dams were selected based on being ± 0.7 standard deviations from the farm’s historical mean litter birth weight for a given litter size and parity. Two male piglets with birth weights close to the mean for their litter of origin were then selected from each HBW and LBW litter. At 4 weeks of age, the piglets were transported from the farm to a biosafety-level 2 animal care facility at the University of Saskatchewan. All piglets were confirmed negative for antibodies to swine influenza A virus prior to transportation. A further RT-PCR assay for influenza was performed on nasal swabs taken after arrival at the facility, and again, all piglets were confirmed negative for the virus. Pigs were acclimated at the facility for 5 days prior to influenza challenge.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from lung tissue from the cranial lobe using the All Prep DNA/RNA mini kit (Qiagen; Hilden, Germany) according to the manufacturer’s recommended protocol. Nucleic acids were quantified by spectrophotometry using a Nanodrop ND 2000 (Thermo Fisher Scientific; Waltham, USA). RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies; Santa Clara, USA). The mean RNA Integrity Number (RIN) sample value was 8.2 ± 0.09. One pig from each sow was randomly selected for microarray analysis, resulting in a total of 32 animals (16 HBW and 16 LBW).
|
| Label |
Cy5
|
| Label protocol |
RNA from the cranial lobe of individual animals was hybridized to separate arrays together with a common reference that included RNA from 3 swine tissues (lung, lymph node, and thymus); a total of 32 hybridizations. A 1 μg amount of total RNA was reverse transcribed into cDNA, which was then used to synthesize antisense RNA (aRNA) by in vitro transcription. Two micrograms of aRNA were then labeled in an ozone-free environment with either Cy3 or Cy5 using the Universal Linkage System (ULS). All these processes used the RNAampULSe kit (Kreatech Diagnostics; Amsterdam, The Netherlands). Labeled aRNAs were purified using the PicoPure RNA Isolation kit (Life Technologies; Carlsbad, USA)
|
| |
|
| Channel 2 |
| Source name |
Pool of Lung, tonsil, and lymph node tissue from 8 HBW and 8 LBW pigs
|
| Organism |
Sus scrofa |
| Characteristics |
age: 4 weeks old
|
| Treatment protocol |
Pigs were inoculated intratracheally with 1x10^7 PFU of SIV strain A/swine/Texas/4199-2/98 H3N2 (TX98) while anaesthetized, and kept in sternal recumbency for 10 minutes. Twice daily during the challenge period, rectal temperatures were taken and the animals were scored for clinical signs of the respiratory disease caused by influenza. Pigs were euthanized 48 hours post-inoculation by pentobarbital injection. Lungs and the attached trachea were removed from the animal and the percentage of lung area affected by consolidation was evaluated and used to select a subset of 'resistant' (RES) and 'susceptible' (SUS) pigs for a separate analysis. Tissue samples from each lobe were then snap-frozen in liquid nitrogen and stored at -80°C for gene expression studies.
|
| Growth protocol |
Litters for the study were selected at birth from a farm in Saskatchewan, Canada. Litter eligibility was based on the average weight of all live and stillborn piglets in the litter. High birth weight (HBW) and low birth weight (LBW) litters from parity 1-2 dams were selected based on being ± 0.7 standard deviations from the farm’s historical mean litter birth weight for a given litter size and parity. Two male piglets with birth weights close to the mean for their litter of origin were then selected from each HBW and LBW litter. At 4 weeks of age, the piglets were transported from the farm to a biosafety-level 2 animal care facility at the University of Saskatchewan. All piglets were confirmed negative for antibodies to swine influenza A virus prior to transportation. A further RT-PCR assay for influenza was performed on nasal swabs taken after arrival at the facility, and again, all piglets were confirmed negative for the virus. Pigs were acclimated at the facility for 5 days prior to influenza challenge.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from lung tissue from the cranial lobe using the All Prep DNA/RNA mini kit (Qiagen; Hilden, Germany) according to the manufacturer’s recommended protocol. Nucleic acids were quantified by spectrophotometry using a Nanodrop ND 2000 (Thermo Fisher Scientific; Waltham, USA). RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies; Santa Clara, USA). The mean RNA Integrity Number (RIN) sample value was 8.2 ± 0.09. One pig from each sow was randomly selected for microarray analysis, resulting in a total of 32 animals (16 HBW and 16 LBW).
|
| Label |
Cy3
|
| Label protocol |
RNA from the cranial lobe of individual animals was hybridized to separate arrays together with a common reference that included RNA from 3 swine tissues (lung, lymph node, and thymus); a total of 32 hybridizations. A 1 μg amount of total RNA was reverse transcribed into cDNA, which was then used to synthesize antisense RNA (aRNA) by in vitro transcription. Two micrograms of aRNA were then labeled in an ozone-free environment with either Cy3 or Cy5 using the Universal Linkage System (ULS). All these processes used the RNAampULSe kit (Kreatech Diagnostics; Amsterdam, The Netherlands). Labeled aRNAs were purified using the PicoPure RNA Isolation kit (Life Technologies; Carlsbad, USA)
|
| |
|
| |
| Hybridization protocol |
825 ng of each dye-labeled aRNA was hybridized for 17 h to Porcine V2 Gene Expression 4x44 Microarrays (Agilent Technologies). Microarray slides were washed using a Gene Expression Wash Pack (Agilent Technologies) according to the manufacturer's reccommended protocols.
|
| Scan protocol |
Hybridization signal intensities were quantified using GenePixPro v7.0 software (Molecular Devices) using the Autoscan function.
|
| Data processing |
Background subtraction was performed on the raw median expression data from each array feature using a ‘norm exp’ method. Next, the background-subtracted expression data were normalized both within array (using a global loess method) and between arrays (using a scale normalization method). The ‘limma simple’ algorithm was then used to assess differential gene expression between the HBW and LBW groups. A fold change threshold of ≥1.75 and a P value of ≤0.05 were set for genes to be classified as differentially expressed. The differential expression of specific genes of interest was validated using an RT-qPCR approach.
|
| |
|
| Submission date |
Oct 28, 2014 |
| Last update date |
Oct 27, 2015 |
| Contact name |
James Michael Wilkinson |
| E-mail(s) |
jamiewilkinson@hotmail.com
|
| Organization name |
University of Alberta
|
| Department |
Agriculture, Food, and Nutritional Science
|
| Street address |
Ag/For Building
|
| City |
Edmonton |
| State/province |
Alberta |
| ZIP/Postal code |
T6G 2P5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL15007 |
| Series (1) |
| GSE62765 |
Effect of litter birth weight phenotype on transcriptomic response to swine influenza virus infection in pig lung |
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