Rubins 2003 - Trizol Extraction of Total RNA from PBMCs; 1. Make sure the area is clean and free of any RNases. Keep the samples cold or on ice as much as possible to prevent RNA degradation. Use only RNase free tubes and RNase free, aerosol-barrier pipette tips throughout the whole experiment. 2. Add 1mL of Trizol to the frozen PBMC pellet. 3. If the Trizol was added to the PBMCs before freezing, thaw the Trizol/PBMCs on the benchtop for 5-10 minutes. 4. Vortex and transfer to 1.5ml microfuge tubes if not already in them. 5. Add 0.2 mls chloroform per 1 ml Trizol. Vortex or shake vigorously and incubate at room temp. for 2-3 minutes. 6. Centrifuge at 12,000g for 15 minutes at 4C. 7. Transfer the aqueous phase to a fresh tube. (The volume will be about 60% of the volume of Trizol.) Note: If isolating DNA, save the organic phase. Add 1uL of 1ug/uL Linear Acrylamide if RNA yield is expected to be low. 8. Add 0.5 mLs of isopropanol per 1 ml Trizol used. Incubate at room temp. for 10 minutes. 9. Centrifuge at top speed (14,000rpm) for 10 minutes at 4C. 10. The RNA pellet will be visible at the bottom of the tube. Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube. 11. WASH 1: Wash the pellet with 1 ml 70% EtOH. Centrifuge at top speed 14,000g for 5-7 minutes. 12. Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube. 13. WASH 2: Wash the pellet with 1 ml 70% EtOH. Centrifuge at top speed 14,000g for 5-7 minutes. 14. Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube. 15. Air dry pellet shortly, until no EtOH is visible (2-5 minutes). Do not overdry! 16. Resuspend the pellet in 20uls of DEPC H2O. Store at ?80C. 17. Check RNA quality and quantity by denaturing gel and OD spec (260nm and 260/280 ratio).; Protocol Type = Extract preparation; Performer: Qing,,Li Rubins 2003 - Ambion Message Amp I; FIRST STRAND CDNA SYNTHESIS: In PCR reaction tube, mix: Amount Reagent 0.1 - 5ug Total RNA 1uL T7 Oligo (dT) primer 11uL Nuclease free water 70oC for 10 min in a thermocycler with lid on, but not clamped down. Centrifuge briefly. Prepare in a Master Mix and keep at room temperature: Amount Reagent 2uL 10X First Strand Buffer 1uL Ribonuclease Inhibitor 4uL dNTP Mix Gently pipette or flick to mix, and then centrifuge briefly. Incubate master mix at 42oC for 2 minutes prior to adding to reaction tubes. Add to the master mix: Amount Reagent 1uL Reverse Transcriptase Quickly transfer 8ul of above Master Mix to each RNA sample, mix by pipetting. 42oC for 2 hours in thermocycler with lid on, but not clamped down. Centrifuge briefly, place on ice, and immediately proceed to next step SECOND STRAND SYNTHESIS: Amount Reagent 63uL Nuclease free water 10uL 10X Second Strand Buffer 4uL dNTP Mix 2uL DNA Polymerase 1uL RNase H Add 80uL to each tube. Gently pipette or flick to mix, and then centrifuge briefly. 16oC for 2 hours in thermocycler with lid completely off. DS cDNA CLEANUP: Preheat the 5mL bottle of water at 50oC for at least 10 minutes. Place filter cartridge in a 2mL tube and add 100ul of cDNA Binding Buffer. Incubate at room temperature for 5 min. Transfer 100uL reaction from strip-8 PCR tubes into 1.5ml Eppendorf tubes. Add 250uL of cDNA binding buffer to each cDNA sample, mix by pipetting. Add the mix to the filter cartridge. Centrifuge for 1 min at 10,000 x g. Discard flow-through. Add 650ul of cDNA wash buffer. Centrifuge for 1 min. at 10,000 x g. Discard flow-through. Centrifuge again for 1 min at 10,000 x g to remove trace amounts of ethanol. Transfer filter cartridge to fresh 2mL tube Add 50uL of preheated water to the center of the column. Incubate at room temperature for 2 minutes. Centrifuge for 1 minute at 10,000 x g. Repeat elution with a second 50ul of preheated water. Centrifuge for 1 minute at 10,000 x g. Transfer to strip-8 PCR tubes and mark a line for 8uL Concentrate the purified cDNA to < 8uL by drying in a vacuum centrifuge on high heat. Resuspend cDNA up to 8uL with H20, mix well, and keep on ice or store at ?20oC. IN VITRO TRANSCRIPTION Prepare the following master mix: Amount Reagent 8uL T7 NTP mix (75 mM each) 2uL T7 10X Reaction Buffer 2uL T7 Enzyme Mix Add 12uL to each tube. Gently pipette or flick to mix, and then centrifuge briefly. 37oC for 6 hours in hybridization oven. Add to each reaction: Amount Reagent 2uL DNaseI Gently pipette or flick to mix, and then centrifuge briefly. 37oC for 30min in hybridization oven aRNA PURIFICATION Preheat 5mL bottle of nuclease free water at 50oC for at least 10 minutes. Immediately before staring aRNA purification., equilibrate 1 filter cartridge per aRNA sample Place filter cartridge in a 2mL tube and add 100ul of aRNA Binding Buffer. Incubate at room temperature for 5 min. Add 78uL aRNA Elution Solution to each IVT mix to bring final volume up to 100uL. Add 350uL of aRNA Binding Buffer to each aRNA sample. Add 250uL of ACS grade 95% ethanol to each sample. Mix by pipetting or gentle vortexing. Apply entire sample to the equilibrated filter cartridge. Centrifuge for 1 minute at 10,000 x g. Discard flow-through but re-use tube. Add 650uL aRNA Wash Buffer to each filter. Centrifuge for 1 minute at 10,000 x g. Discard flow-through but re-use tube. Centrifuge again for 1 min at 10,000 x g Transfer cartridge to a fresh 2mL tube. Elute aRNA by adding 50uL preheated (50oC) water to center of column. Incubate at room temperature for 2 min. Centrifuge for 1 minute at 10,000 x g. Repeat elution with a second 50uL of preheated water. Centrifuge for 1 minute at 10,000 x g. Store aRNA at ?80oC.; Protocol Type = Amplification; Performer: Qing,,Li
Label
Cy3
Label protocol
Rubins 2003 - Labeling Amplified RNA - Direct Incorporation of Cy Dyes; Preparation of Fluorescent DNA Probe from Amplified mRNA 1. To anneal primer, mix 3-4ug of amplified mRNA with 10ug of random hexamer primer in a total volume of 15.4ul: mRNA 3-4ug pdN6 (5 ug/uL) 2uL ddH2O (DEPC) to 15.4 uL Total volume: 15.4 uL 2. Heat to 65oC for 10 min and cool on ice. 3. Add 14.6uL reaction mixture (Master Mix or separate) to each RNA/hexamer mix Reaction mixture 5X first-strand buffer 6.0uL 0.1M DTT 3.0uL Unlabeled dNTPs (low dTTP) 0.6uL Cy3 or Cy5 (1 mM, Amersham) 3.0uL Superscript II (200 U/uL, Gibco BRL) 2.0uL Total volume: 14.6uL 4. Incubate at 42oC for 1 hr. 5. Add 1 uL SSII (RT booster) to each sample. Incubate for an additional 1 hr. 6. Degrade RNA by addition 15uL of 0.1N NaOH and incubate at 65-70oC for 10 min. 7. Neutralize by addition of 15uL of 0.1N HCl. 8. Add 380uL of TE (10mM Tris, 1mM EDTA) to a Microcon YM-30 column. Next add the 60uL of Cy5 probe and the 60??l of Cy3 probe to the same Microcon. 9. WASH 1: Spin 7-8 min. at 14,000g (=RCF). Remove flow through and save to a separate tube. 10. WASH 2: Add 450ul 1X TE and spin 7-8 min. at 14,000g. Remove flow through and save to the separate tube. 11. WASH 3: Add to centricon: 1X TE 450uL 1ug/uL Cot1 human DNA (Gibco-BRL) 20 uL 10ug/uL polyA RNA (Sigma, #P9403) 2 uL 10ug/uL tRNA (Gibco-BRL, #15401-011) 2 uL Spin 7-10 min. at 14,000g. Look for nice concentration of the "colored probe" in the centricon. Try to concentrate to a volume of less than 25uL. This volume is attained when the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane. Make sure not to dry the membrane completely! 12. Invert the centricon into a clean tube and spin briefly at 14,000 RPM to recover the probe.; Protocol Type = Labeling; Performer: Qing,,Li
cell type: BDC2.5 6wk_1 old Pancreatic LN CD4 cells
Extracted molecule
total RNA
Extraction protocol
Rubins 2003 - Trizol Extraction of Total RNA from PBMCs; 1. Make sure the area is clean and free of any RNases. Keep the samples cold or on ice as much as possible to prevent RNA degradation. Use only RNase free tubes and RNase free, aerosol-barrier pipette tips throughout the whole experiment. 2. Add 1mL of Trizol to the frozen PBMC pellet. 3. If the Trizol was added to the PBMCs before freezing, thaw the Trizol/PBMCs on the benchtop for 5-10 minutes. 4. Vortex and transfer to 1.5ml microfuge tubes if not already in them. 5. Add 0.2 mls chloroform per 1 ml Trizol. Vortex or shake vigorously and incubate at room temp. for 2-3 minutes. 6. Centrifuge at 12,000g for 15 minutes at 4C. 7. Transfer the aqueous phase to a fresh tube. (The volume will be about 60% of the volume of Trizol.) Note: If isolating DNA, save the organic phase. Add 1uL of 1ug/uL Linear Acrylamide if RNA yield is expected to be low. 8. Add 0.5 mLs of isopropanol per 1 ml Trizol used. Incubate at room temp. for 10 minutes. 9. Centrifuge at top speed (14,000rpm) for 10 minutes at 4C. 10. The RNA pellet will be visible at the bottom of the tube. Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube. 11. WASH 1: Wash the pellet with 1 ml 70% EtOH. Centrifuge at top speed 14,000g for 5-7 minutes. 12. Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube. 13. WASH 2: Wash the pellet with 1 ml 70% EtOH. Centrifuge at top speed 14,000g for 5-7 minutes. 14. Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube. 15. Air dry pellet shortly, until no EtOH is visible (2-5 minutes). Do not overdry! 16. Resuspend the pellet in 20uls of DEPC H2O. Store at ?80C. 17. Check RNA quality and quantity by denaturing gel and OD spec (260nm and 260/280 ratio).; Protocol Type = Extract preparation; Performer: Qing,,Li Rubins 2003 - Ambion Message Amp I; FIRST STRAND CDNA SYNTHESIS: In PCR reaction tube, mix: Amount Reagent 0.1 - 5ug Total RNA 1uL T7 Oligo (dT) primer 11uL Nuclease free water 70oC for 10 min in a thermocycler with lid on, but not clamped down. Centrifuge briefly. Prepare in a Master Mix and keep at room temperature: Amount Reagent 2uL 10X First Strand Buffer 1uL Ribonuclease Inhibitor 4uL dNTP Mix Gently pipette or flick to mix, and then centrifuge briefly. Incubate master mix at 42oC for 2 minutes prior to adding to reaction tubes. Add to the master mix: Amount Reagent 1uL Reverse Transcriptase Quickly transfer 8ul of above Master Mix to each RNA sample, mix by pipetting. 42oC for 2 hours in thermocycler with lid on, but not clamped down. Centrifuge briefly, place on ice, and immediately proceed to next step SECOND STRAND SYNTHESIS: Amount Reagent 63uL Nuclease free water 10uL 10X Second Strand Buffer 4uL dNTP Mix 2uL DNA Polymerase 1uL RNase H Add 80uL to each tube. Gently pipette or flick to mix, and then centrifuge briefly. 16oC for 2 hours in thermocycler with lid completely off. DS cDNA CLEANUP: Preheat the 5mL bottle of water at 50oC for at least 10 minutes. Place filter cartridge in a 2mL tube and add 100ul of cDNA Binding Buffer. Incubate at room temperature for 5 min. Transfer 100uL reaction from strip-8 PCR tubes into 1.5ml Eppendorf tubes. Add 250uL of cDNA binding buffer to each cDNA sample, mix by pipetting. Add the mix to the filter cartridge. Centrifuge for 1 min at 10,000 x g. Discard flow-through. Add 650ul of cDNA wash buffer. Centrifuge for 1 min. at 10,000 x g. Discard flow-through. Centrifuge again for 1 min at 10,000 x g to remove trace amounts of ethanol. Transfer filter cartridge to fresh 2mL tube Add 50uL of preheated water to the center of the column. Incubate at room temperature for 2 minutes. Centrifuge for 1 minute at 10,000 x g. Repeat elution with a second 50ul of preheated water. Centrifuge for 1 minute at 10,000 x g. Transfer to strip-8 PCR tubes and mark a line for 8uL Concentrate the purified cDNA to < 8uL by drying in a vacuum centrifuge on high heat. Resuspend cDNA up to 8uL with H20, mix well, and keep on ice or store at ?20oC. IN VITRO TRANSCRIPTION Prepare the following master mix: Amount Reagent 8uL T7 NTP mix (75 mM each) 2uL T7 10X Reaction Buffer 2uL T7 Enzyme Mix Add 12uL to each tube. Gently pipette or flick to mix, and then centrifuge briefly. 37oC for 6 hours in hybridization oven. Add to each reaction: Amount Reagent 2uL DNaseI Gently pipette or flick to mix, and then centrifuge briefly. 37oC for 30min in hybridization oven aRNA PURIFICATION Preheat 5mL bottle of nuclease free water at 50oC for at least 10 minutes. Immediately before staring aRNA purification., equilibrate 1 filter cartridge per aRNA sample Place filter cartridge in a 2mL tube and add 100ul of aRNA Binding Buffer. Incubate at room temperature for 5 min. Add 78uL aRNA Elution Solution to each IVT mix to bring final volume up to 100uL. Add 350uL of aRNA Binding Buffer to each aRNA sample. Add 250uL of ACS grade 95% ethanol to each sample. Mix by pipetting or gentle vortexing. Apply entire sample to the equilibrated filter cartridge. Centrifuge for 1 minute at 10,000 x g. Discard flow-through but re-use tube. Add 650uL aRNA Wash Buffer to each filter. Centrifuge for 1 minute at 10,000 x g. Discard flow-through but re-use tube. Centrifuge again for 1 min at 10,000 x g Transfer cartridge to a fresh 2mL tube. Elute aRNA by adding 50uL preheated (50oC) water to center of column. Incubate at room temperature for 2 min. Centrifuge for 1 minute at 10,000 x g. Repeat elution with a second 50uL of preheated water. Centrifuge for 1 minute at 10,000 x g. Store aRNA at ?80oC.; Protocol Type = Amplification; Performer: Qing,,Li
Label
Cy5
Label protocol
Rubins 2003 - Labeling Amplified RNA - Direct Incorporation of Cy Dyes; Preparation of Fluorescent DNA Probe from Amplified mRNA 1. To anneal primer, mix 3-4ug of amplified mRNA with 10ug of random hexamer primer in a total volume of 15.4ul: mRNA 3-4ug pdN6 (5 ug/uL) 2uL ddH2O (DEPC) to 15.4 uL Total volume: 15.4 uL 2. Heat to 65oC for 10 min and cool on ice. 3. Add 14.6uL reaction mixture (Master Mix or separate) to each RNA/hexamer mix Reaction mixture 5X first-strand buffer 6.0uL 0.1M DTT 3.0uL Unlabeled dNTPs (low dTTP) 0.6uL Cy3 or Cy5 (1 mM, Amersham) 3.0uL Superscript II (200 U/uL, Gibco BRL) 2.0uL Total volume: 14.6uL 4. Incubate at 42oC for 1 hr. 5. Add 1 uL SSII (RT booster) to each sample. Incubate for an additional 1 hr. 6. Degrade RNA by addition 15uL of 0.1N NaOH and incubate at 65-70oC for 10 min. 7. Neutralize by addition of 15uL of 0.1N HCl. 8. Add 380uL of TE (10mM Tris, 1mM EDTA) to a Microcon YM-30 column. Next add the 60uL of Cy5 probe and the 60??l of Cy3 probe to the same Microcon. 9. WASH 1: Spin 7-8 min. at 14,000g (=RCF). Remove flow through and save to a separate tube. 10. WASH 2: Add 450ul 1X TE and spin 7-8 min. at 14,000g. Remove flow through and save to the separate tube. 11. WASH 3: Add to centricon: 1X TE 450uL 1ug/uL Cot1 human DNA (Gibco-BRL) 20 uL 10ug/uL polyA RNA (Sigma, #P9403) 2 uL 10ug/uL tRNA (Gibco-BRL, #15401-011) 2 uL Spin 7-10 min. at 14,000g. Look for nice concentration of the "colored probe" in the centricon. Try to concentrate to a volume of less than 25uL. This volume is attained when the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane. Make sure not to dry the membrane completely! 12. Invert the centricon into a clean tube and spin briefly at 14,000 RPM to recover the probe.; Protocol Type = Labeling; Performer: Qing,,Li
Hybridization protocol
not provided
Scan protocol
Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Datafile type = GenePix Results 3; Parameter Image Origin = 1160, 5120; Software: type: feature extraction; Performer: Qing,,Li Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scan Date = 2006-04-26; Parameter Scanning software = AxImageIO; Parameter Pixel Size = 10; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter PMT Volts = 590; Parameter Laser on time = 291500; Parameter Laser Power = 1.40; Parameter Scan Time = 13:01:59; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scanner Model = GenePix 4000A [47263]; Parameter Focus Position = 0; Parameter Scan Temperature = 39.22; Performer: Qing,,Li Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scan Date = 2006-04-26; Parameter Scanning software = AxImageIO; Parameter Scanning software version = 2.0.2.3; Parameter Pixel Size = 10; Parameter PMT Volts = 560; Parameter Scan Power = 100; Parameter Laser on time = 656410; Parameter Laser Power = 2.08; Parameter Scan Time = 13:01:59; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scanner Model = GenePix 4000A [47263]; Parameter Focus Position = 0; Parameter Scan Temperature = 39.22; Performer: Qing,,Li
IFN-alpha induced gene expression profile in NOD/BDC2.5 and NOD mice.
Data table header descriptions
ID_REF
ID_REF
CH1I_MEAN
Mean feature pixel intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale
CH2I_MEAN
Mean feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale
CH1B_MEDIAN
The median feature background intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel; Background
CH2B_MEDIAN
The median feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background
CH1D_MEAN
The mean feature pixel intensity at wavelength 532 nm with the median background subtracted.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel
CH2D_MEAN
.The mean feature pixel intensity at wavelength 635 nm with the median background subtracted.; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH1I_MEDIAN
Median feature pixel intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale
CH2I_MEDIAN
Median feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale
CH1B_MEAN
The mean feature background intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Background
CH2B_MEAN
The mean feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Background
CH1D_MEDIAN
The median feature pixel intensity at wavelength 532 nm with the median background subtracted.; Type: integer; Scale: linear_scale
CH2D_MEDIAN
The median feature pixel intensity at wavelength 635 nm with the median background subtracted.; Type: integer; Scale: linear_scale
CH1_PER_SAT
The percentage of feature pixels at wavelength 532 nm that are saturated.; Type: integer; Scale: linear_scale
CH2_PER_SAT
The percentage of feature pixels at wavelength 635 nm that are saturated.; Type: integer; Scale: linear_scale
CH1I_SD
The standard deviation of the feature intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel
CH2I_SD
The standard deviation of the feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH1B_SD
The standard deviation of the feature background intensity at wavelength 532 nm.; Type: float; Scale: linear_scale; Channel: Cy3 Channel; Background
CH2B_SD
The standard deviation of the feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background
PERGTBCH1I_1SD
The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale
PERGTBCH2I_1SD
The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 635 nm.; Type: integer; Scale: linear_scale
PERGTBCH1I_2SD
The percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale
PERGTBCH2I_2SD
The percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale
SUM_MEAN
The sum of the arithmetic mean intensities for each wavelength, with the median background subtracted.; Type: integer; Scale: linear_scale
SUM_MEDIAN
The sum of the median intensities for each wavelength, with the median background subtracted.; Type: integer; Scale: linear_scale
RAT1_MEAN
Ratio of the arithmetic mean intensities of each spot for each wavelength, with the median background subtracted. Channel 1/Channel 2 ratio, (CH1I_MEAN - CH1B_MEDIAN)/(CH2I_MEAN - CH2B_MEDIAN) or Green/Red ratio.; Type: float; Scale: linear_scale
RAT2_MEAN
The ratio of the arithmetic mean intensities of each feature for each wavelength, with the median background subtracted.; Type: float; Scale: linear_scale
RAT2_MEDIAN
The ratio of the median intensities of each feature for each wavelength, with the median background subtracted.; Type: float; Scale: linear_scale
PIX_RAT2_MEAN
The geometric mean of the pixel-by-pixel ratios of pixel intensities, with the median background subtracted.; Type: float; Scale: linear_scale
PIX_RAT2_MEDIAN
The median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted.; Type: float; Scale: linear_scale
RAT2_SD
The geometric standard deviation of the pixel intensity ratios.; Type: float; Scale: linear_scale
TOT_SPIX
The total number of feature pixels.; Type: integer; Scale: linear_scale
TOT_BPIX
The total number of background pixels.; Type: integer; Scale: linear_scale
REGR
The regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature.; Type: float; Scale: linear_scale
CORR
The correlation between channel1 (Cy3) & Channel 2 (Cy5) pixels within the spot, and is a useful quality control parameter. Generally, high values imply better fit & good spot quality.; Type: float; Scale: linear_scale
DIAMETER
The diameter in um of the feature-indicator.; Type: integer; Scale: linear_scale
X_COORD
X-coordinate of the center of the spot-indicator associated with the spot, where (0,0) is the top left of the image.; Type: integer; Scale: linear_scale
Y_COORD
Y-coordinate of the center of the spot-indicator associated with the spot, where (0,0) is the top left of the image.; Type: integer; Scale: linear_scale
The type of flag associated with a feature: -100 = user-flagged null spot; -50 = software-flagged null spot; 0 = spot valid.; Type: integer; Scale: linear_scale
CH2IN_MEAN
Normalized value of mean Channel 2 (usually 635 nm) intensity (CH2I_MEAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH2BN_MEDIAN
Normalized value of median Channel 2 (usually 635 nm) background (CH2B_MEDIAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background
CH2DN_MEAN
Normalized value of mean Channel 2 (usually 635 nm) intensity with normalized background subtracted (CH2IN_MEAN - CH2BN_MEDIAN).; Type: integer; Scale: linear_scale; Channel: Cy5 channel
RAT2N_MEAN
Type: float; Scale: linear_scale
CH2IN_MEDIAN
Normalized value of median Channel 2 (usually 635 nm) intensity (CH2I_MEDIAN/Normalization factor).; Type: integer; Scale: linear_scale
CH2DN_MEDIAN
Normalized value of median Channel 2 (usually 635 nm) intensity with normalized background subtracted (CH2IN_MEDIAN - CH2BN_MEDIAN).; Type: integer; Scale: linear_scale
RAT1N_MEAN
Ratio of the means of Channel 1 (usually 532 nm) intensity to normalized Channel 2 (usually 635 nm) intensity with median background subtracted (CH1D_MEAN/CH2DN_MEAN). Channel 1/Channel 2 ratio normalized or Green/Red ratio normalized.; Type: float; Scale: linear_scale
RAT2N_MEDIAN
Channel 2/Channel 1 ratio normalized, RAT2_MEDIAN/Normalization factor or Red/Green median ratio normalized.; Type: float; Scale: linear_scale
LOG_RAT2N_MEAN
Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm) [log (base 2) (RAT2N_MEAN)].; Type: float; Scale: log_base_2
VALUE
Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm) [log (base 2) (RAT2N_MEDIAN)].; Type: float; Scale: log_base_2
