|
| Status |
Public on Dec 31, 2020 |
| Title |
Listeria monocytogenes_48C_v 2.0_biological3_tech rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Listeria monocytogenes
|
| Organism |
Listeria monocytogenes |
| Characteristics |
strain: ATCC 43256
treatment group: control
|
| Biomaterial provider |
PFGRC
|
| Treatment protocol |
Overnight cultures of L. monocytogenes were inoculated in fresh LB broth and the strain was incubated at 37°C with agitation at 100 rpm until OD600 0.5 (~ 108 CFU/mL) was reached (control).
|
| Growth protocol |
Overnight cultures of L. monocytogenes were inoculated in fresh LB broth and the strain was incubated at 37°C with agitation at 100 rpm until OD600 0.5 (~ 108 CFU/mL) was reached. At this point, aliquots were placed into multiple 1.5 mL microfuge tubes for the different heat treatments. Heat treatments were performed in a calibrated water-bath (BOEKEL Grant ORS200, PA, USA) using temperature probes that facilitated temperature monitoring within the sample contained in the microfuge tubes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was isolated from the samples using an RNeasy™ midi kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. RNAprotect™ bacteria reagent (Qiagen) was added to the cultures to stabilize RNA before the isolation. The RNase-free DNase set (Qiagen) was used for on-column DNA digestion to remove residual genomic DNA. The quantity and quality of RNA was examined using the NanoDrop (ND-1000) spectrophotometer (NanoDrop® Technologies, Palo Alto, CA), and the Bioanalyzer 2100™ (Agilent Technologies, Santa Clara, CA), respectively.
|
| Label |
Cy5
|
| Label protocol |
Total RNA (10 µg) was used to synthesize cDNA using a random primer for reverse transcription (Invitrogen Life Technologies, Carlsbad, CA). The primer was annealed at 70°C for 10 min, followed by snap-freezing in a dry ice-ethanol bath for 30 s and centrifugation for 1 min. The reaction mixture (Superscript III buffer, 0.1 M dithiothreitol, and aminoallyl-deoxyuridine triphosphate mix) was then incubated overnight with Superscript III reverse transcriptase (Invitrogen) at 42°C. Residual RNA was removed by alkaline treatment followed by neutralization, and the cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified cDNAs from the mutant was labeled with Cy-5 mono-Reactive Dye (GE Health Care).
|
| |
|
| Channel 2 |
| Source name |
Listeria monocytogenes
|
| Organism |
Listeria monocytogenes |
| Characteristics |
strain: ATCC 43256
treatment group: thermo-tolerance
|
| Biomaterial provider |
PFGRC
|
| Treatment protocol |
Cells were incubated at 48°C for 30 minutes to induce thermo-tolerance.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was isolated from the samples using an RNeasy™ midi kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. RNAprotect™ bacteria reagent (Qiagen) was added to the cultures to stabilize RNA before the isolation. The RNase-free DNase set (Qiagen) was used for on-column DNA digestion to remove residual genomic DNA. The quantity and quality of RNA was examined using the NanoDrop (ND-1000) spectrophotometer (NanoDrop® Technologies, Palo Alto, CA), and the Bioanalyzer 2100™ (Agilent Technologies, Santa Clara, CA), respectively.
|
| Label |
Cy3
|
| Label protocol |
Total RNA (10 µg) was used to synthesize cDNA using a random primer for reverse transcription (Invitrogen Life Technologies, Carlsbad, CA). The primer was annealed at 70°C for 10 min, followed by snap-freezing in a dry ice-ethanol bath for 30 s and centrifugation for 1 min. The reaction mixture (Superscript III buffer, 0.1 M dithiothreitol, and aminoallyl-deoxyuridine triphosphate mix) was then incubated overnight with Superscript III reverse transcriptase (Invitrogen) at 42°C. Residual RNA was removed by alkaline treatment followed by neutralization, and the cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified cDNAs from the mutant was labeled with Cy-5 mono-Reactive Dye (GE Health Care).
|
| |
|
| |
| Hybridization protocol |
The labeling mixtures were further purified using a QIAquick PCR purification kit (Qiagen). Equal amounts of labeled cDNA from the treatment and control were used to hybridize on L. monocytogenes genome microarrays [version 2 arrays developed by JCVI and provided by the Pathogen Functional Genome Resource Center (PFGRC) of the National Institutes of Health]. These arrays were 70 mer-oligo arrays with 6347 oligonucleotides each, with 4 replicate spots per oligonucleotides. The labeled cDNA was applied to the above arrays. Hybridization was carried out overnight at 42°C in a water bath using Corning hybridization chamber. The pre-and post-hybridization was carried out using the the TIGR SOP #M008.
|
| Scan protocol |
The slides was washed and scanned using a GenePix 4100A scanner (Axon Instruments Inc., Union City, CA) at 532 nm (Cy3 channel) and 635 nm (Cy5 channel), and the image was stored for further analysis.
|
| Description |
The slide was pre-hybridized following the TIGR SOP # M007
|
| Data processing |
The signal intensity of each gene was globally normalized using LOWESS within the R statistics package. (Normalizationfunctions.RData and R.2.5.1).
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| |
|
| Submission date |
Jan 11, 2011 |
| Last update date |
Dec 31, 2020 |
| Contact name |
Palmy R Jesudhasan |
| E-mail(s) |
palmy.jesudhasan@utsouthwestern.edu
|
| Phone |
214-648-5627
|
| Organization name |
University of Texas Southwestern Medical Center
|
| Department |
Microbiology
|
| Street address |
NL4.110M
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
| |
|
| Platform ID |
GPL4284 |
| Series (1) |
| GSE26570 |
Differential expression of genes in Listeria monocytogenes under thermo-tolerance condition of heat shock |
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