|
| Status |
Public on Oct 28, 2005 |
| Title |
E12.5 Embryo1, 5 pooled(16011321000038) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
E12.5 Embryos, 5 pooled
|
| Organism |
Mus musculus |
| Characteristics |
mixed C57BL/6 mixed sex embryo
|
| Biomaterial provider |
NIA Animal Resources Facility
|
| Treatment protocol |
Pregnant female mice from timed matings were sacrificed by cervical dislocation at 12.5 days post-coitus. Decidua were removed to 1x PBS on ice, and embryos/placentas were dissected and transferred to iced PBS. For each litter, embryos were combined in one tube, while placentas were combined in another. Tubes were snap frozen on dry ice and stored at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TROZOL standard extraction method. The full protocol can be found in the Life Technologies CAT 15596. The following are the steps we used. 1.Homogenize tissue samples in 1 mL of TRIZOL Reagent per 50-100 mg of tissue using a glass-Teflon® or power homogenizer (Polytron, or Tekmar's TISSUMIZER® or equivalent). The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization. 3.PHASE SEPARATION Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 mL of chloroform per 1 mL of TRIZOL Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 2 to 8°C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization. 4. RNA PRECIPITATION Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 mL of isopropyl alcohol per 1 mL of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes at 2 to 8°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 5. RNA WASH Remove the supernate. Wash the RNA pellet once with 75% ethanol, adding at least 1 mL of 75% ethanol per 1 mL of TRIZOL Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at 2 to 8°C. 6. REDISSOLVING THE RNA At the end of the procedure, briefly dry the RNA pellet (ai
|
| Label |
Cy3
|
| |
|
| Channel 2 |
| Source name |
E12.5 Embryos, 5 pooled
|
| Organism |
Mus musculus |
| Characteristics |
mixed C57BL/6 mixed sex embryo
|
| Biomaterial provider |
NIA Animal Resources Facility
|
| Treatment protocol |
Pregnant female mice from timed matings were sacrificed by cervical dislocation at 12.5 days post-coitus. Decidua were removed to 1x PBS on ice, and embryos/placentas were dissected and transferred to iced PBS. For each litter, embryos were combined in one tube, while placentas were combined in another. Tubes were snap frozen on dry ice and stored at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TROZOL standard extraction method. The full protocol can be found in the Life Technologies CAT 15596. The following are the steps we used. 1.Homogenize tissue samples in 1 mL of TRIZOL Reagent per 50-100 mg of tissue using a glass-Teflon® or power homogenizer (Polytron, or Tekmar's TISSUMIZER® or equivalent). The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization. 3.PHASE SEPARATION Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 mL of chloroform per 1 mL of TRIZOL Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 2 to 8°C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization. 4. RNA PRECIPITATION Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 mL of isopropyl alcohol per 1 mL of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes at 2 to 8°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 5. RNA WASH Remove the supernate. Wash the RNA pellet once with 75% ethanol, adding at least 1 mL of 75% ethanol per 1 mL of TRIZOL Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at 2 to 8°C. 6. REDISSOLVING THE RNA At the end of the procedure, briefly dry the RNA pellet (ai
|
| Label |
Cy5
|
| |
|
| |
| Hybridization protocol |
Slides were hybridized according to manufacturers protocol (Agilent in situ Microarray Hybridization Protocol, Product # G2559A or P/N G2556-66003, September 10, 2001)
|
| Description |
E12.5 Embryos, 5 pooled
|
| Data processing |
Data are extracted with Agilent Feature Extraction Software.The data were further processed with NIA ANOVA tool utilities.See http://lgsun.grc.nia.nih.gov/ANOVA for details.
|
| |
|
| Submission date |
Oct 25, 2005 |
| Last update date |
Oct 27, 2005 |
| Contact name |
Minoru S.H. Ko |
| E-mail(s) |
[email protected]
|
| Phone |
410-558-8359
|
| Organization name |
NIH
|
| Department |
National Institute on Aging
|
| Lab |
Lab of Genetics
|
| Street address |
251 Bayview Blvd, Suite 100, 10C
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL870 |
| Series (2) |
| GSE3507 |
In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling: Part 3 |
| GSE3513 |
In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling: Part2 |
|
| Data table header descriptions |
| ID_REF |
Feature number |
| PositionX |
Found X coordinate of feature centroid in pixels |
| PositionY |
Found Y coordinate of feature centroid in pixels |
| VALUE |
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. A feature is considered found if the found spot centroid is within the bounds of the spot deviation limit with respect to corresponding nominal centroid. |
| rIsFound |
A boolean used to flag found (strong) features.The flag is applied independently in each channel. A feature is considered found if the found spot centroid is within the bounds of the spot deviation limit with respect to corresponding nominal centroid. |
| gProcessedSignal |
Dye-normalized signal after surrogate algorithm, green channel, used for computation of log ratio |
| rProcessedSignal |
Dye-normalized signal after surrogate algorithm, red channel, used for computation of log ratio |
| gProcessedSigError |
Standard error of propagated feature signal, green channel |
| rProcessedSigError |
Standard error of propagated feature signal, red channel |
| gNumPixOLHi |
Number of outlier pixels per feature with intensity > upper threshold set via the pixel outlier rejection method. The number is computed independently in each channel. These pixels are omitted from all subsequent calculations. |
| rNumPixOLHi |
Number of outlier pixels per feature with intensity > upper threshold set via the pixel outlier rejection method. The number is computed independently in each channel. These pixels are omitted from all subsequent calculations. |
| gNumPixOLLo |
Number of outlier pixels per feature with intensity < lower threshold set via the pixel outlier rejection method. The number is computed independently in each channel. NOTE: The pixel outlier method is the ONLY step that removes data in Feature Extraction. |
| rNumPixOLLo |
Number of outlier pixels per feature with intensity < lower threshold set via the pixel outlier rejection method. The number is computed independently in each channel. NOTE: The pixel outlier method is the ONLY step that removes data in Feature Extraction. |
| 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 |
Raw median signal of feature in green channel (inlier pixels) |
| rMedianSignal |
Raw median signal of feature in red 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 |
| rIsSaturated |
Boolean flag indicating if a feature is saturated or not |
| PixCorrelation |
Ratio of estimated feature covariance in RedGreen space to product of feature Standard Deviation in Red Green space. |
| BGPixCorrelation |
Ratio of estimated background covariance in RedGreen space to product of background Standard Deviation in Red Green space. |
| gIsFeatNonUnifOL |
Boolean flag indicating if a feature is NonUniformity Outlier or not Green Channel |
| rIsFeatNonUnifOL |
Boolean flag indicating if a feature is NonUniformity Outlier or not Red Channel |
| gIsBGNonUnifOL |
Boolean flag indicating if background is NonUniformity Outlier or not Green Channel |
| rIsBGNonUnifOL |
Boolean flag indicating if background is NonUniformity Outlier or not Red Channel |
| gIsFeatPopnOL |
Boolean flag indicating if a feature is a Population Outlier or not for Green Channel. 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 for Red Channe. Probes with replicate features on a microarray are examined using population statistics |
| gIsBGPopnOL |
Boolean flag indicating if background is a Population Outlier or not for Green Channel. |
| rIsBGPopnOL |
Boolean flag indicating if background is a Population Outlier or not for Red Channel. |
| IsManualFlag |
Manual Flag |
| gBGSubSignal |
The net g signal following the subtraction of the background from the raw mean g signal |
| rBGSubSignal |
The net r signal following the subtraction of the background from the raw mean r signal |
| gBGSubSigError |
Propagated standard error as computed on net g background subtracted signal |
| rBGSubSigError |
Propagated standard error as computed on net r background subtracted signal |
| BGSubSigCorrelation |
Ratio of estimated background subtracted feature signal covariance in RG space to product of background subtracted feature Standard Deviation in RG space |
| gIsPosAndSignif |
Boolean flag indicating 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 indicating 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 Mean signal and g background (selected by user) |
| rPValFeatEqBG |
P-value from t-test of significance between r Mean signal and r background (selected by user) |
| gNumBGUsed |
Number of local background regions or features used to calculate the background subtraction on this feature g channel. |
| rNumBGUsed |
Number of local background regions or features used to calculate the background subtraction on this feature r channel. |
| gIsWellAboveBG |
Boolean flag indicating if a feature is WellAbove Background or not |
| rIsWellAboveBG |
Boolean flag indicating if a feature is WellAbove Background or not |
| IsUsedBGAdjust |
A boolean used to flag features used for computation of global BG offset |
| gBGUsed |
Background value (after global background adjust if turned ON) subtracted from the raw mean signal to generate the BG subtracted signal; this value is computed per channel. If global BG subtraction is used the column is identical for every feature in a given channel |
| rBGUsed |
Background value (after global background adjust if turned ON) subtracted from the raw mean signal to generate the BG subtracted signal; this value is computed per channel. If global BG subtraction is used the column is identical for every feature in a given channel |
| gBGSDUsed |
Standard deviation of background used in g channel |
| rBGSDUsed |
Standard deviation of background used in r channel |
| IsNormalization |
A 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 |
