Number of individuals: 1; mean weight: 55.3 g; Tissue: hematopoietic end of the kidney (the anterior 1/3rd including the head and mid kidney)
Treatment protocol
The same procedure as challenged samples was followed for the fish in a control tank except they were exposed to 100 ml of Hank’s balanced salt solution.
Extracted molecule
total RNA
Extraction protocol
The -80oC samples were removed and processed immediately by adding 0.75 ml of Trizol (Invitrogen) and one stainless steel 3 mm ball. The tubes were placed in a Retsch mixer mix for two cycles of 3 min/20Hz. Chloroform (0.3 ml) was added to the resulting homogenate and vortexed for 30 sec. The phases were separated by centrifugation at 13000 rpm at 5 ºC for 15 min. The aqueous phase was transferred to a tube containing 0.2 ml isopropanol and mixed. The RNA was precipitated for 5 min and then centrifuged as before. The total RNA pellet was washed with 80% cold ethanol, centrifuged, and incubated with 1 U of DNase for 15 min followed by gel electrophoresis and spectrophotometric analysis for quality and quantity.
Label
Alexa 555
Label protocol
Twenty micrograms of total RNA were reverse transcribed into cDNA and labelled with Alexa dyes using the Invitrogen Indirect Labelling Kit, with modifications from the manufacturer’s instructions.
Channel 2
Source name
pooled reference sample from all individuals collected
Number of individuals: 72; mean weight: 55.3 g; Tissue: hematopoietic end of the kidney (the anterior 1/3rd including the head and mid kidney)
Treatment protocol
challenged individuals were exposed to 100 ml of IHN, control individuals were exposed to100 ml of Hank’s balanced salt solution.
Extracted molecule
total RNA
Extraction protocol
kidney tissue from each individual was extracted separately as described for channel 1 sample. Equal quantities of RNA from each sample were mixed for the pooled sample.
Label
Alexa 647
Label protocol
the same as channel 1 sample
Hybridization protocol
The microarray slides were prepared by washing in 0.01% SDS and 3 water washes, followed by a denaturation step at 80 oC for 3 min. The slides were then directly placed into a pre-hybridization solution (5X SSC, 0.01% SDS; 0.2% BSA) V) at 43 oC for 1h. The slides were washed 3 times in water and centrifuged dry using 50 ml falcon tubes and a bench top centrifuge (1600xg). The slides were assembled into the Corning slide chambers and 3X SSC were added to the reservoirs. The denatured targets (sample and pooled reference) were added to the slides and slide covers and chambers were assembled immediately. The slides were incubated for 16 hr at 45 oC. Slides were then washed at low (0.01% SDS; 1xSSC) and high (0.1X SSC) stringency, centrifuged to dry.
Scan protocol
All arrarys were scanned using a Perkin Elmer ScanArray Express (Perkin Elmer, Boston, MA) , adjusting the PMT gain for optimized visualization of each image.
Description
kidney tissue from a single fish exposed to the IHN virus in a waterborne challenge compared to a major reference pool of kidney from all collected samples, treated and untreated (n=72).
Data processing
The images were quantified using Imagene (BioDiscovery, El Segundo, CA, www.biodiscovery.com) and processed to GeneSight (version 4.1, BioDiscovery, Inc. El Segundo, CA, www.biodiscovery.com). Data was prepared in GeneSight as following steps: correct for background intensity using local background correction; omit multiple spots with flags(1,2,3,4,5,6,7); raise all values below a specified value to the minimum threshold (20.0), replace missing values by the experimental means for each gene, LOWESS normalization is done locally with smoothing parameter of 0.2 and linear degree of fitness, finally calculate the log base 2 and the difference of the log2 of 2 channels. This pairMean log difference (VALUE) was used for all subsequent data analysis.
Salmonid host response to Hematopoietic Necrosis virus: cellular receptors, viral control and novel pathways of defence
Data table header descriptions
ID_REF
VALUE
same as UNF_VALUE but with flagged values removed
CH1_Flag
Channel 1 Numeric code of the flag for the spot (0=no flag, 1=flag spots by manual flags, 2=empty spots by automatic flags, 3=poor spots by automatic flags, 4=negative spots by automatic flags, 5=empty spots by manual flags, 6=poor spots by manual flags, 7=negative spots by manual flags. )
CH1_Signal_Mean
Channel 1 Pixel intensity averaged over the local signal region
CH1_Background_Mean
Channel 1 Pixel intensity averaged over the local background region
CH1_Signal_Median
Channel 1 Median pixel intensity computed over the local signal region
CH1_Background_Median
Channel 1 Median pixel intensity computed over the local background region
CH1_Signal_Stdev
Channel 1 Standard deviation of pixel intensities over the local signal region
CH1_Background_Stdev
Channel 1 Standard deviation of pixel intensities over the local background region
CH1_Shape_Regularity
Channel 1 First signal area of a spot is inscribed into a circle. Then number of non-signal pixels that fall within this circle is computed and divided by circle's area. This ratio is subtracted from 1 as is called shape regularity
CH1_Spot_Area
Channel 1 Signal Area plus Ignored Area
CH2_Flag
Channel 2 Numeric code of the flag for the spot (0=no flag, 1=flag spots by manual flags, 2=empty spots by automatic flags, 3=poor spots by automatic flags, 4=negative spots by automatic flags, 5=empty spots by manual flags, 6=poor spots by manual flags, 7=negative spots by manual flags. )
CH2_Signal_Mean
Channel 2 Pixel intensity averaged over the local signal region
CH2_Background_Mean
Channel 2 Pixel intensity averaged over the local background region
CH2_Signal_Median
Channel 2 Median pixel intensity computed over the local signal region
CH2_Background_Median
Channel 2 Median pixel intensity computed over the local background region
CH2_Signal_Stdev
Channel 2 Standard deviation of pixel intensities over the local signal region
CH2_Background_Stdev
Channel 2 Standard deviation of pixel intensities over the local background region
CH2_Shape_Regularity
Channel 2 First signal area of a spot is inscribed into a circle. Then number of non-signal pixels that fall within this circle is computed and divided by circle's area. This ratio is subtracted from 1 as is called shape regularity
