|
| Status |
Public on Oct 01, 2012 |
| Title |
Intestinal vs. control SmedArray2-Biorep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control
|
| Organism |
Schmidtea mediterranea |
| Characteristics |
cell type: non-intestinal cells (column flow-through)
strain: asexual
|
| Treatment protocol |
One hundred large planarians were fed a mixture of liver homogenate, food coloring, and Feridex (AMAG Pharmaceuticals). Forty-eight hours later, animals were dissociated into single cell suspensions essentially as described (Reddien et al., 2005), with the exception that dispase (Worthington Biochemical, final 0.6U/mL) was substituted for trypsin. Intestinal phagocytes were purified on Miltenyi magnetic columns.
|
| Growth protocol |
Asexual Schmidtea mediterranea were maintained in 1X Montjuic salts at 21°C (Cebrià et al., 2005)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All intestinal cells (2-5 x 10^5) and ~10% of non-intestinal cells (1-3 x 10^6) were lysed in Trizol (Invitrogen). RNA was isolated according to the manufacturerʼs instructions, using the high-salt step for RNA precipitation. RNA was DNAse-treated, purified, and concentrated using the DNA-free RNA kit (Zymo Research). 500 ng of total RNA were amplified using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion).
|
| Label |
Alexa Fluor 647
|
| Label protocol |
~12 μg aminoallyl-labeled aRNA were coupled to Alexa Fluor 555 or Alexa Fluor 647 succinimidyl ester dyes (Molecular Probes) and purified according to the MessageAmp II protocol. Labeling efficiency was calculated using a NanoDrop spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Intestine
|
| Organism |
Schmidtea mediterranea |
| Characteristics |
cell type: Intestinal phagocytes
strain: asexual
|
| Treatment protocol |
One hundred large planarians were fed a mixture of liver homogenate, food coloring, and Feridex (AMAG Pharmaceuticals). Forty-eight hours later, animals were dissociated into single cell suspensions essentially as described (Reddien et al., 2005), with the exception that dispase (Worthington Biochemical, final 0.6U/mL) was substituted for trypsin. Intestinal phagocytes were purified on Miltenyi magnetic columns.
|
| Growth protocol |
Asexual Schmidtea mediterranea were maintained in 1X Montjuic salts at 21°C (Cebrià et al., 2005)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All intestinal cells (2-5 x 10^5) and ~10% of non-intestinal cells (1-3 x 10^6) were lysed in Trizol (Invitrogen). RNA was isolated according to the manufacturerʼs instructions, using the high-salt step for RNA precipitation. RNA was DNAse-treated, purified, and concentrated using the DNA-free RNA kit (Zymo Research). 500 ng of total RNA were amplified using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion).
|
| Label |
Alexa Fluor 555
|
| Label protocol |
~12 μg aminoallyl-labeled aRNA were coupled to Alexa Fluor 555 or Alexa Fluor 647 succinimidyl ester dyes (Molecular Probes) and purified according to the MessageAmp II protocol. Labeling efficiency was calculated using a NanoDrop spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
Labeled aRNA targets were hydrolyzed using RNA Fragmentation Reagents (Ambion/Applied Biosystems) according to the manufacturerʼs instructions. Combimatrix arrays were hydrated, prehybridized, hybridized, and washed according to the manufacturerʼs protocols.
|
| Scan protocol |
Arrays were covered with Imaging Solution and a LifterSlip, and imaged using a GenePix 4000B Scanner and GenePix Pro 6.0 software (Axon Instruments/Molecular Devices).
|
| Description |
This is the second of four biological replicates used in this experiment, each prepared separately.
|
| Data processing |
GenePix “.gpr” files were processed using the limma package in Bioconductor (Gentleman et al., 2004; Smyth, 2004). Within-array loess normalization was conducted with the normexp background correction method (Ritchie et al., 2007) and an offset value of 25; the scale method was utilized for between-array normalization. Normalized values for each spot were fitted to a linear model, and moderated t-statistics and adjusted p-values were calculated for each probe on the array.
|
| |
|
| Submission date |
Feb 06, 2012 |
| Last update date |
Oct 01, 2012 |
| Contact name |
Noelle James |
| Organization name |
U of IL / HHMI
|
| Department |
Cell and Developmental Biology
|
| Lab |
Newmark Lab
|
| Street address |
601 S. Goodwin Ave.
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL15193 |
| Series (1) |
| GSE35565 |
Expression analysis of purified intestinal phagocytes in Schmidtea mediterranea |
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