|
| Status |
Public on Sep 06, 2010 |
| Title |
Patient 3, replicate |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
in vitro
|
| Organism |
Salmonella enterica subsp. enterica serovar Paratyphi A |
| Characteristics |
serovar: Paratyphi A
growth condition: in vitro (LB media)
patient: 3
|
| Growth protocol |
To create cDNA of organisms in the blood of bacteremic patients (in vivo samples), we used TRIzol-preserved blood samples of patients whose day 0 culture subsequently grew S. Paratyphi A. To generate corresponding in vitro cDNA samples, we grew each patient's bacterial isolate in Luria Bertani (LB) broth until mid-log growth phase (OD600 0.45-0.6), and then immediately placed these samples into TRIzol at a 1 (mid-log culture):2 (TRIzol) volume ratio.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
We recovered total RNA from TRIzol-preserved in vivo and in vitro samples per manufacturer's instructions (Invitrogen), and treated with DNase on RNeasy columns (Qiagen). We converted 5 µg of total extracted RNA into cDNA using random priming (T-PCR) to obtain a representative cDNA population as described by Froussard (Froussard P. 1992. Nucleic Acids Res 20: 2900 [PMID 1614887]), with modifications as previously described by Graham (Graham JE. 1999. PNAS. 96: 11554-11559 [PMID 10500215]). We performed three rounds of SCOTS on in vivo and in vitro cDNA samples, as previously described (Graham JE. 1999. PNAS. 96: 11554-11559 [PMID 10500215]).
|
| Label |
Cy3
|
| Label protocol |
We differently labeled in vivo and in vitro SCOTS-cDNAs for each of three patients with S. Paratyphi A bacteremia (Dziejman et. al. (2002) PNAS, 99: 1556-1561 [PMID 11818571]).
|
| |
|
| Channel 2 |
| Source name |
in vivo
|
| Organism |
Salmonella enterica subsp. enterica serovar Paratyphi A |
| Characteristics |
serovar: Paratyphi A
growth condition: in vivo (blood of bacteremic patient)
patient: 3
|
| Growth protocol |
To create cDNA of organisms in the blood of bacteremic patients (in vivo samples), we used TRIzol-preserved blood samples of patients whose day 0 culture subsequently grew S. Paratyphi A. To generate corresponding in vitro cDNA samples, we grew each patient's bacterial isolate in Luria Bertani (LB) broth until mid-log growth phase (OD600 0.45-0.6), and then immediately placed these samples into TRIzol at a 1 (mid-log culture):2 (TRIzol) volume ratio.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
We recovered total RNA from TRIzol-preserved in vivo and in vitro samples per manufacturer's instructions (Invitrogen), and treated with DNase on RNeasy columns (Qiagen). We converted 5 µg of total extracted RNA into cDNA using random priming (T-PCR) to obtain a representative cDNA population as described by Froussard (Froussard P. 1992. Nucleic Acids Res 20: 2900 [PMID 1614887]), with modifications as previously described by Graham (Graham JE. 1999. PNAS. 96: 11554-11559 [PMID 10500215]). We performed three rounds of SCOTS on in vivo and in vitro cDNA samples, as previously described (Graham JE. 1999. PNAS. 96: 11554-11559 [PMID 10500215]).
|
| Label |
Cy5
|
| Label protocol |
We differently labeled in vivo and in vitro SCOTS-cDNAs for each of three patients with S. Paratyphi A bacteremia (Dziejman et. al. (2002) PNAS, 99: 1556-1561 [PMID 11818571]).
|
| |
|
| |
| Hybridization protocol |
Labeled products were added to activated Salmonella ORF microarray glass slides (version STv7S; McClelland Laboratory, Vaccine Research Institute of San Diego, CA, http://www.sdibr.org/Faculty/mcclelland/mcclelland-lab), and developed as previously described (Larocque RC et. al. (2005). Infect Immun 73: 4488-4493 [PMID 16040959]).
|
| Scan protocol |
Arrays were scanned using a Perkin Elmer scanner and ScanArray Express software version 4.
|
| Description |
Analysis used in vitro RNA as control samples for comparison to the in vivo samples.
|
| Data processing |
For analyses, we subtracted local background from spot signal intensities, and considered a cDNA for an ORF detected in a particular sample if it met the following criteria: 1) the median signal intensity of at least 2 of its 3 replicate spots on the array was at least ten median absolute deviations greater than the median of spots on the microarray corresponding to genes absent from the S. Paratyphi A genome, and 2) this criteria was met on greater than 75% of slides for that subject. We then evaluated differences in expression in in vitro versus in vivo grown organisms for all genes detected in in vivo samples. Using LOESS-normalized, log-transformed data, we employed repeated measures ANOVA (to within slide replicate spots) with fixed type (in vivo versus in vitro) and dye effects with Benjamini-Hochberg correction. We only considered array features with a coefficient of variation in signal intensity of less than 50% within an array. We considered significant variations in signal intensity as determined by ANOVA indication of potentially differentially expressed genes. Since the samples were hybridized to two-channel arrays, the data from different slides were not normalized to have the same median intensities. Instead we used a simple (standard) method of analysis described by Kerr et. al (2001) Genetics Research, 77:123-128 (PMID 11355567) which included a slide effect in the analysis of variance model. Thus, for each gene when comparing in vivo and in vitro levels, the slide effect was adjusted for in the model fit, and there was no need to normalize data to have same median intensities across the slides.
|
| |
|
| Submission date |
Sep 05, 2010 |
| Last update date |
Sep 05, 2010 |
| Contact name |
Richelle C. Charles |
| E-mail(s) |
rccharles@partners.org
|
| Organization name |
Massachussetts General Hospital
|
| Department |
Infectious Diseases
|
| Lab |
Ryan
|
| Street address |
55 Fruit Street, Gray 504
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL10883 |
| Series (1) |
| GSE22958 |
High-throughput gene expression profiling of Salmonella enterica serovar Paratyphi A in the blood of bacteremic patients in Bangladesh |
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