|
| Status |
Public on May 10, 2007 |
| Title |
Tcl1 target genes in embryonic stem cells, biological replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Tcl1 stable transformant, clone 1
|
| Organism |
Mus musculus |
| Characteristics |
EB3 origin, genotype:(Tcl1-/-,CAG-Tcl1)
|
| Growth protocol |
Embryonic stem cells were routinely cultured without feeder cells in GMEM supplemented with 10% fetal calf serum, 100microM 2-mercaptoethanol, non-essential amino acid, sodium pyruvate and LIF on gelatin-coated dishes. All assays were carried out before passage 30.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the acid guanidinium-phenol-chloroform (AGPC) method. The denaturing solution was added directly to cell culture dishes washed 3 times with PBS. The quality of purified total RNA was examined by Agilent 2100 Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
Low RNA Input Fluorescent Linear Amplification Kit(Agilent) was utilized to obtain Cyanine5 labelled cRNA from 500ng total RNA. The cRNA was purified with a RNeasy mini spin column(QIAGEN). Then 0.75microg each of Cyanine3 and Cyanine5 labelled cRNAs were combined, fragmented and used for hybridization to the microarray.
|
| |
|
| Channel 2 |
| Source name |
Tcl1 gene disruptant, clone 4
|
| Organism |
Mus musculus |
| Characteristics |
EB3 origin, genotype:Tcl1-/-
|
| Growth protocol |
Embryonic stem cells were routinely cultured without feeder cells in GMEM supplemented with 10% fetal calf serum, 100microM 2-mercaptoethanol, non-essential amino acid, sodium pyruvate and LIF on gelatin-coated dishes. All assays were carried out before passage 30.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the acid guanidinium-phenol-chloroform (AGPC) method. The denaturing solution was added directly to cell culture dishes washed 3 times with PBS. The quality of purified total RNA was examined by Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Low RNA Input Fluorescent Linear Amplification Kit(Agilent) was utilized to obtain Cyanine3 labelled cRNA from 500ng total RNA. The cRNA was purified with a RNeasy mini spin column(QIAGEN). Then 0.75microg each of Cyanine3 and Cyanine5 labelled cRNAs were combined, fragmented and used for hybridization to the microarray.
|
| |
|
| |
| Description |
A targeting vector replacing parts of exon 2 and exon3 with a PGK-puro cassette was used to disrupt the Tcl1 locus. Clones homozygous for the disrupted Tcl locus were isolated by selecting Tcl1+/- cells with high concentration puromycin. We next transfected Tcl-/- clone 4 with the CAG-Tcl1ireszeopA expression vector.
|
| Data processing |
The data processing to obtain the VALUE is as follows. 1. Yield the background-subtracted intensity for each feature. 2. Correct dye biases. 3. In situations when the raw mean signal is less than th background or not signifiicant compared to the background, or the background-subtracted signal is less than its backround standard deviation, the signal is replaced with a surrogate signal. 4. The log ratio of the red over green processed signals is calculated. See Agilent Feature Extraction Software Reference Guide for details.
|
| |
|
| Submission date |
May 10, 2006 |
| Last update date |
May 10, 2006 |
| Contact name |
Tatsushi Miyazaki |
| E-mail(s) |
tatsu@nutri.med.osaka-u.ac.jp
|
| Organization name |
Osaka University Graduate School of Medicine
|
| Department |
Department of Molecular Therapeutics
|
| Lab |
Stem Cell Regulation Research
|
| Street address |
2-2 Yamadaoka
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE4801 |
Comparative profiling of Tcl1 gene disruptants and Tcl1 stable transformants. |
|
| Data table header descriptions |
| ID_REF |
Agilent feature number |
| VALUE |
(base 10) log(REDsignal/GREENsignal) per feature (processed signals used) |
| LogRatioError |
Error of the log ratio calculated according to the error model chosen |
| PValueLogRatio |
Significance level of the Log Ratio computed for a feature |
| gSurrogateUsed |
The green surrogate value used |
| rSurrogateUsed |
The red surrogate value used |
| gIsFound |
A boolean used to flag found (strong) features. The flag is applied independently in each channel |
| rIsFound |
A boolean used to flag found (strong) features. The flag is applied independently in each channel |
| gProcessedSignal |
Dye-normalized signal after surrogate algorithm, per channel, used for computation of log ratio |
| rProcessedSignal |
Dye-normalized signal after surrogate algorithm, per channel, used for computation of log ratio |
| gProcessedSigError |
Standard error of propagated feature signal, per channel |
| rProcessedSigError |
Standard error of propagated feature signal, per channel |
| gNumPixOLHi |
Number of outlier pixels per feature with intensity > upper threshold set via the pixel outlier rejection method |
| rNumPixOLHi |
Number of outlier pixels per feature with intensity > upper threshold set via the pixel outlier rejection method |
| gNumPixOLLo |
Number of outlier pixels per feature with intensity < lower threshold set via the pixel outlier rejection method |
| rNumPixOLLo |
Number of outlier pixels per feature with intensity < lower threshold set via the pixel outlier rejection method |
| gNumPix |
Total number of pixels used to compute feature statistics; ie. total number of inlier pixels/per spot; same in both channels |
| rNumPix |
Total number of pixels used to compute feature statistics; ie. total number of inlier pixels/per spot; same in both channels |
| gMeanSignal |
Raw mean signal of feature in green channel (inlier pixels) |
| rMeanSignal |
Raw mean signal of feature in red channel (inlier pixels) |
| gMedianSignal |
Median local background signal (local to corresponding feature) computed per channel (inlier pixels) |
| rMedianSignal |
Median local background signal (local to corresponding feature) computed per channel (inlier pixels) |
| gPixSDev |
Standard deviation of all inlier pixels per feature; this is computed independently in each channel |
| rPixSDev |
Standard deviation of all inlier pixels per feature; this is computed independently in each channel |
| gBGNumPix |
Total number of pixels used to compute local BG statistics per spot; ie. total number of BG inlier pixels; same in both channels |
| rBGNumPix |
Total number of pixels used to compute local BG statistics per spot; ie. total number of BG inlier pixels; same in both channels |
| gBGMeanSignal |
Mean local background signal (local to corresponding feature) computed per channel (inlier pixels) |
| rBGMeanSignal |
Mean local background signal (local to corresponding feature) computed per channel (inlier pixels) |
| gBGMedianSignal |
Median local background signal (local to corresponding feature) computed per channel (inlier pixels) |
| rBGMedianSignal |
Median local background signal (local to corresponding feature) computed per channel (inlier pixels) |
| gBGPixSDev |
Standard deviation of all inlier pixels per local BG of each feature, computed independently in each channel |
| rBGPixSDev |
Standard deviation of all inlier pixels per local BG of each feature, computed independently in each channel |
| gNumSatPix |
Total number of saturated pixels per feature, computed per channel |
| rNumSatPix |
Total number of saturated pixels per feature, computed per channel |
| gIsSaturated |
Boolean flag indicating if a feature is saturated or not. A feature is saturated IF 50% of the pixels in a feature are above the saturation threshold |
| rIsSaturated |
Boolean flag indicating if a feature is saturated or not. A feature is saturated IF 50% of the pixels in a feature are above the saturation threshold |
| PixCorrelation |
Ratio of estimated feature covariance in RedGreen space to product of feature Standard Deviation in Red Green space |
| BGPixCorrelation |
The same concept as above but in case of background |
| gIsFeatNonUnifOL |
Boolean flag indicating if a feature is a NonUniformity Outlier or not. A feature is non-uniform if the pixel noise of feature exceeds a threshold established for a "uniform" feature |
| rIsFeatNonUnifOL |
Boolean flag indicating if a feature is a NonUniformity Outlier or not. A feature is non-uniform if the pixel noise of feature exceeds a threshold established for a "uniform" feature |
| gIsBGNonUnifOL |
The same concept as above but in case of background |
| rIsBGNonUnifOL |
The same concept as above but in case of background |
| gIsFeatPopnOL |
Boolean flag indicating if a feature is a Population Outlier or not. Probes with replicate features on a microarray are examined using population statistics |
| rIsFeatPopnOL |
Boolean flag indicating if a feature is a Population Outlier or not. Probes with replicate features on a microarray are examined using population statistics |
| gIsBGPopnOL |
The same concept as above but in case of background |
| rIsBGPopnOL |
The same concept as above but in case of background |
| IsManualFlag |
Boolean is manual flag |
| gBGSubSignal |
Background-subtracted signal |
| rBGSubSignal |
Background-subtracted signal |
| gBGSubSigError |
Propagated standard error as computed on net g(r) background subtracted signal |
| rBGSubSigError |
Propagated standard error as computed on net g(r) background subtracted signal |
| BGSubSigCorrelation |
Ratio of estimated background-subtracted feature signal co-variance in RG space to product of background-subtracted feature Standard Deviation in RG space |
| gIsPosAndSignif |
Boolean flag, established via a 2-sided t-test, indicates if the mean signal of a feature is greater than the corresponding background (selected by user) and if this difference is significant |
| rIsPosAndSignif |
Boolean flag, established via a 2-sided t-test, indicates if the mean signal of a feature is greater than the corresponding background (selected by user) and if this difference is significant |
| gPValFeatEqBG |
P-value from t-test of significance between g(r) Mean signal and g(r) background (selected by user) |
| rPValFeatEqBG |
P-value from t-test of significance between g(r) Mean signal and g(r) background (selected by user) |
| gNumBGUsed |
Background used to subtract from the MeanSignal; variable also used in t-test |
| rNumBGUsed |
Background used to subtract from the MeanSignal; variable also used in t-test |
| gIsWellAboveBG |
Boolean flag indicating if a feature is WellAbove Background or not |
| rIsWellAboveBG |
Boolean flag indicating if a feature is WellAbove Background or not |
| gBGUsed |
Background used to subtract fromthe MeanSignal; variable also used in t-test |
| rBGUsed |
Background used to subtract fromthe MeanSignal; variable also used in t-test |
| gBGSDUsed |
Standard deviation of background used in g(r) channel; variable also used in t-test and surrogate algorithms |
| rBGSDUsed |
Standard deviation of background used in g(r) channel; variable also used in t-test and surrogate algorithms |
| IsNormalization |
Boolean flag which indicates if a feature is used to measure dye bias |
| gDyeNormSignal |
The dye-normalized signal in the indicated channel |
| rDyeNormSignal |
The dye-normalized signal in the indicated channel |
| gDyeNormError |
The standard error associated with the dye-normalized signal |
| rDyeNormError |
The standard error associated with the dye-normalized signal |
| DyeNormCorrelation |
Dye-normalized red and green pixel correlation |
| ErrorModel |
Indicates the error model that you chose for feature extraction or that the software uses if you have chosen the "most conservative" option |
| IsUsedBGAdjust |
Boolean used to flag features used for computation of global BG offset |
