|
| Status |
Public on Dec 31, 2011 |
| Title |
HT-29 human cells challenged 3h with Lactobacillus plantarum 299v |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HT-29 + Lactobacillus plantarum 299v
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HT-29 intestinal epithelial cells (IEC)
treatment: stimulated with a 100: 1 bateria to cell ratio of L. plantarum 299v
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by TRIzol Reagent (Invitrogen, Burlington, ON, Canada) following manufacturer's protocol and with PhaseLock tubes (5 Prime, Gaithersburg, MD) for nucleic acids separation. Purification was achieved with RNeasy (QIAGEN, Germantown, MD). Quality was assessed by Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada). RIN over 9.0 were considered good quality RNA for reverse transcription.
|
| Label |
Cy3,Cy5
|
| Label protocol |
Poly A mRNA were reverse transcribed from 15 µg of total RNA with SuperScript III (Invitrogen) and labelled with Cy3 (GE Healthcare Life Sciences, Buckinghamshire, England) before cDNA purification with PCR Purification Kit (QIAGEN).
|
| |
|
| Channel 2 |
| Source name |
Control (HT-29 cells only)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HT-29 intestinal epithelial cells (IEC)
treatment: not stimulated with anything (bacteria to cell ration of 0: 1)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by TRIzol Reagent (Invitrogen, Burlington, ON, Canada) following manufacturer's protocol and with PhaseLock tubes (5 Prime, Gaithersburg, MD) for nucleic acids separation. Purification was achieved with RNeasy (QIAGEN, Germantown, MD). Quality was assessed by Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada). RIN over 9.0 were considered good quality RNA for reverse transcription.
|
| Label |
Cy5,Cy3
|
| Label protocol |
Poly A mRNA were reverse transcribed from 15 µg of total RNA with SuperScript III (Invitrogen) and labelled with Cy5 (GE Healthcare Life Sciences, Buckinghamshire, England) before cDNA purification with PCR Purification Kit (QIAGEN).
|
| |
|
| |
| Hybridization protocol |
Cy3 and Cy5 labelled cDNA were mixed and hybridized at 50 Celcius degrees during 18h on the Immune Array chips in an ArrayBooster (Advalytix, Beckman Coulter, Germany).
|
| Scan protocol |
Each array was scanned in a ScanArray 5000 instrument (Perkin Elmer, Waltham, MA) and acquired images were quantified with QuantArray software (Perkin Elmer).
|
| Data processing |
Statistical analyses were done with R software (v. 2.13.0) and the Limma package from Bioconductor. Normalization within slide was obtained by LOWESS correction and between slides normalization was done with Aquantile method.
|
| |
|
| Submission date |
Aug 15, 2011 |
| Last update date |
Dec 31, 2011 |
| Contact name |
Thomas Allan Tompkins |
| Organization name |
Lallemand Health Solutions Inc.
|
| Department |
R&D
|
| Street address |
6100 Royalmount Avenue
|
| City |
Montreal |
| State/province |
Qc |
| ZIP/Postal code |
H4P 2R2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13933 |
| Series (1) |
| GSE31394 |
Transcriptomic response of immune signaling pathways in intestinal epithelial cells exposed to commensals, pathogens and probiotics |
|
