Ti: RNA derived from chicken trachea tissues at day i (i=1,3,5,8,12,21,22,24) after the initial IBV-Mass inoculation; Ci: Control RNA derived from chicken trachea tissue at day i after inoculation with sterile distilled water. Note: Chickens were re-inoculated with IBV-Mass at day 21 after the initial immunization. We used pooled control RNA for hybridization.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the pooled samples of age-matched control and vaccinated chickens using RNeasy mini tissue kit from QIAGEN Inc. (Valencia, CA). Residue DNA was removed with RNase-free DNase (QIAGEN). The concentration of total RNA was determined using NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). The quality and quantity of selected RNA samples was examined by gel electrophoresis.
Label
Cy3
Label protocol
Message RNA amplification was carried out with MessageAmp aminoallyl Amplification Kit (Ambion, #1752). Then, amplified RNA was used for cDNA synthesis, which was subsequently labeled with Cy3 and Cy5 Monoreactive dye (Amersham, # PA23001 and # PA25001). Purification of the cDNA was performed with QIAquick PCR Purification Kit (QIAGEN, #28104). All procedures were performed by following the manufacturers’ instructions.
Ti: RNA derived from chicken trachea tissues at day i (i=1,3,5,8,12,21,22,24) after the initial IBV-Mass inoculation; Ci: Control RNA derived from chicken trachea tissue at day i after inoculation with sterile distilled water. Note: Chickens were re-inoculated with IBV-Mass at day 21 after the initial immunization. We used pooled control RNA for hybridization.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the pooled samples of age-matched control and vaccinated chickens using RNeasy mini tissue kit from QIAGEN Inc. (Valencia, CA). Residue DNA was removed with RNase-free DNase (QIAGEN). The concentration of total RNA was determined using NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). The quality and quantity of selected RNA samples was examined by gel electrophoresis.
Label
Cy5
Label protocol
Message RNA amplification was carried out with MessageAmp aminoallyl Amplification Kit (Ambion, #1752). Then, amplified RNA was used for cDNA synthesis, which was subsequently labeled with Cy3 and Cy5 Monoreactive dye (Amersham, # PA23001 and # PA25001). Purification of the cDNA was performed with QIAquick PCR Purification Kit (QIAGEN, #28104). All procedures were performed by following the manufacturers’ instructions.
Hybridization protocol
Hybridization was performed based on the protocol provided by FHCRC (http://www.fhcrc.org/science/shared_resources/genomics/dna_array/spotted_arrays/chicken_array/HybridizationWashProtocol.pdf) with slight modifications. Briefly, nonfat milk was used in pre-hybridization to block non-specific binding sites. For hybridization, the mixture of 20X SSC (6.0µl), 2% SDS (2.4µl) and labeled cDNA samples (5µg for each) was applied to 13k array slide. The total volume was 60µl. Slide was incubated at 63°C for 4hrs followed by incubating at 55°C for 11hrs.
Scan protocol
After washing, slides were dried by quick spin and ready for scanning. Arrays were scanned using a GenePix 4000B Microarray Scanner (Molecular Device Corporation, Sunnyvale, CA) and image analysis was performed by using GenePixPro6.0.
Description
The goal of the study was to uncover the molecular mechanism of mucosal immunity development using avian infectious bronchitis virus (IBV) as a model system. To achieve this goal, we monitored the kinetics of local gene transcription profiles in trachea tissues after administration of animals with an attenuated IBV strain (IBV-Mass) using chicken 13K cDNA Microarray. More specifically, immune-related gene transcription profiles in trachea at 1, 3, 5, 8, 12 and 21 days after the primary immunization and at 1 and 2 days after a second immunization were characterized. There are total 9 groups including 8 time points for vaccinated groups and 1 pooled age-matched control group. RNAs from each group were used to compare with RNAs from other four groups as determined by the loop-design. Four different RNA samples at each time point were used for hybridization with the respective four other groups. The use of the loop-design allows the direct comparisons between the 9 groups, which would enhance the statistical power and lower the variation. This design is preferred over other methods when the goal of the study is to uncover the kinetics of gene transcription profiles.
Data processing
Data were collected using GenePixPro6.0 with the LOWESS normalization.
