|
| Status |
Public on Apr 12, 2011 |
| Title |
Mussel hemolymph_ 48 h post V. splendidus LGP32 injection_48.2 arrayB |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Hemolymph, control
|
| Organism |
Mytilus galloprovincialis |
| Characteristics |
biological replica: Control pool (N=40)
tissue: hemolymph
treatment: 0.1 ml PBS-NaCl injection into the posterior adductor muscle
time point: 48 h post injection
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer's instructions, and further purified by LiCl precipitation.
|
| Label |
Cy3
|
| Label protocol |
Hemolymph mRNA was linearly amplified from total RNA with the Ambion Message-AmpTM II aRNA Amplification kit. Initially, modified nucleotides 5-(3-aminoallyl)-UTP were incorporated into the aRNA during the in vitro transcription reaction; then, mono-functional NHS-esters of Cy3 or -Cy5 dyes (CyDye Post-Labeling Reactive Dye Pack, Amersham GE Healthcare) were resuspended in DMSO and covalently coupled to the aminoallyl-aRNA probes for 1 h at room temperature in the dark.
|
| |
|
| Channel 2 |
| Source name |
Hemolymph h 48.2_B
|
| Organism |
Mytilus galloprovincialis |
| Characteristics |
biological replica: Pool 2 (N=10)
tissue: hemolymph
treatment: 10 to 7 CFU of V. splendidus LGP32 injection into the posterior adductor muscle
time point: 48 h post injection
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer's instructions, and further purified by LiCl precipitation.
|
| Label |
Cy5
|
| Label protocol |
Hemolymph mRNA was linearly amplified from total RNA with the Ambion Message-AmpTM II aRNA Amplification kit. Initially, modified nucleotides 5-(3-aminoallyl)-UTP were incorporated into the aRNA during the in vitro transcription reaction; then, mono-functional NHS-esters of Cy3 or -Cy5 dyes (CyDye Post-Labeling Reactive Dye Pack, Amersham GE Healthcare) were resuspended in DMSO and covalently coupled to the aminoallyl-aRNA probes for 1 h at room temperature in the dark.
|
| |
|
| |
| Hybridization protocol |
Following purification (Gene Elute PCR Clean-up kit, Sigma-Aldrich) and UV-quantification, 500 ng of both reference and test aaRNAs were combined and ethanol-precipitated. Cy3/Cy5-coupled samples were re-suspended in 18 µl of hybridization buffer (5x SSC, 50% formamide, 0.1% SDS), denaturated for 3 min at 70°C and competitively hybridised to the Immunochip for 24 h at 48°C in humidified dual-slide chamber (HybChamber, GeneMachines). Slides were preliminary conditioned for 12 h at 48°C in a solution of 5x SSC, 100 ng/µl salmon sperm ssDNA, 5x Denhardt’s solution and 0.1% SDS); then, reference and test samples were simultaneously hybridised in dye-swap crossed combinations on the two identical arrays of the same slide. The slides were sequentially washed at room temperature with mild shaking in buffer: 1x SSC, 0.2% SDS; 0.1x SSC, 0.2% SDS; 0.2x SSC (4 min each) and 0.1x SSC (3 min), with final drying by air flow.
|
| Scan protocol |
Immunochip fluorescence signals were scanned using two lasers (633 nm and 543 nm) at 5 μm resolution with a GSI Lumonics LITE dual confocal laser scanner.
|
| Description |
Image processing and signal quantification were performed with ScanArray Express® (PerkinElmer) whose output files report spot data, pixel statistics, quality parameters and the fluorescence intensity values for each probe and channel.
|
| Data processing |
Normalisation of the fluorescence signals was performed by using the total and LOWESS (Logfit) algorithm with MIDAS (MIcroarray Data Analysis System, http://www.tigr.org/software). The log2 test/reference ratio of all the normalised fluorescence values was computed.
|
| |
|
| Submission date |
Aug 10, 2010 |
| Last update date |
Apr 12, 2011 |
| Contact name |
Laura Varotto |
| E-mail(s) |
lauravarotto@yahoo.it
|
| Phone |
00390498276284
|
| Organization name |
University of Padova
|
| Department |
Biology Department
|
| Street address |
via U. Bassi 58/3
|
| City |
Padova |
| ZIP/Postal code |
35121 |
| Country |
Italy |
| |
|
| Platform ID |
GPL10758 |
| Series (1) |
| GSE23535 |
Performance of Immunochip 1.0 with hemolymph samples collected at 3 and 48 hours from Vibrio-challenged mussels |
|
