|
| Status |
Public on Nov 20, 2006 |
| Title |
infected HCT116 tumor replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
infected HCT116 tumor
|
| Organisms |
Homo sapiens; Clostridium novyi NT |
| Characteristics |
infected HCT116 tumor
|
| Growth protocol |
Six- to 8-week-old athymic nu/nu mice (Harlan, Indianapolis, IN) were inoculated subcutaneously with five million HCT116 human cancer cells. Three hundred million C. novyi-NT spores were administered by tail-vein injection once the tumor volumes reached ~500 mm3. Germination was generally observed to initiate within 12 hours and RNA isolated eighteen hours later, just after germination became evident.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Vegetative bacteria or spores were collected by centrifugation and homogenized by vortexing in RNAwiz buffer (RiboPureTM-Bacteria kit, Ambion, Austin, TX) containing 250 µL of Zirconia beads (Biospec, Bartlesville, OK) for 10 minutes at 4°C, followed by a 5-min break. This cycle was repeated 12 times to ensure disruption of the spores (as assessed by phase contrast microscopy and RNA yield). RNA was purified with the RiboPureTM-Bacteria kit (Ambion) following the manufacturer’s instructions.
RNA was prepared from infected or uninfected tumors by homogenizing (50 strokes) the dissected tumors in a glass homogenizer containing a 10-fold volume of the RNAwiz buffer on ice. The homogenate was aliquoted into 0.5-mL RNase-free tubes. The samples were then processed exactly as described above, using a 12-cycle homogenization with Zirconia beads.
|
| Label |
biotin
|
| Label protocol |
total RNA was reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen). The first-strand cDNA was fragmented by DNase I (Promega, Madison WI) and labeled with biotinylated ddATP through a terminal transferase end-labeling reaction.
|
| |
|
| Hybridization protocol |
Hybridization with the end-labeled samples was carried out at 45°C for 16 hours in 100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20 and 10.5% glycerol.
|
| Scan protocol |
After washing, the arrays were stained with Cy3-Streptavidin, followed by scanning with an Axon 4000B scanner.
|
| Description |
The features were extracted by using the NimbleScan software.
|
| Data processing |
Data was analyzed with the Bioconductor project using quantile normalization (see:Bolstad B, Irizarry R, Astrand M, Speed T (2003) A comparison of normalization methods for high density oligonucleotide array data based on bias and variance. Bioinformatics 19 (2), p185-193) and gene calls generated using the RMA algorithm (Robust multichip average described in Biostatistics 2003 Apr;4(2):249-64 ).
|
| |
|
| Submission date |
Oct 19, 2006 |
| Last update date |
Nov 20, 2006 |
| Contact name |
Xin Huang |
| E-mail(s) |
xhuang8@jhmi.edu
|
| Organization name |
Johns Hopkins University
|
| Department |
Oncology
|
| Street address |
1650 Orleans Street, CRB I, Room 520
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL4463 |
| Series (1) |
| GSE6087 |
Transcriptomes of Clostridium novyi-NT |
|
