|
| Status |
Public on Jan 26, 2011 |
| Title |
A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Listeria monocytogenes EGD
|
| Organism |
Listeria monocytogenes |
| Characteristics |
strain: EGD
genotype/variation: wild type
|
| Growth protocol |
Strains were grown to early stationary phase in BHI broth at 37C, defined as OD 1 + 3h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the RNeasy mini or midi kit from Qiagen as described by the manufacturer. Cells grown to early-stationary phase were first treated with RNA protect as instructed by the manufacturer (Qiagen) and subsequently disrupted by sonication on ice (3 X 30 seconds, each round followed by a 30 second pause). RNA was then extracted using the Qiagen RNeasy kit. The integrity of the RNA was confirmed by agarose gel electrophoresis and the concentration and purity was determined on a NanoDrop 2000.
|
| Label |
Cy3 or Cy5
|
| Label protocol |
cDNA from each strain was labeled twice with Cy3 and twice with Cy5 to minimize any bias. cDNA synthesis and labeling of total RNA were performed using the SuperScript Plus indirect cDNA labeling system for DNA microarrays (Invitrogen). 10 µg total RNA was mixed with 5 µg random hexamers and incubated for 10 minutes at 70ºC, with a subsequent chill on ice for at least 5 minutes. Superscript III RT, amino-modified deoxynucleoside triphophates, dithiothreitol, RNaseOUT, and buffer was then added and the reaction mix incubated at 42ºC for 17 hours. RNA was hydrolyzed by the addition of 10 µl 1M NaOH and 10 µl 0.5 M EDTA, followed by incubation at 65ºC for 15 minutes. The mixture was neutralized with 10 µl 1M HCl and cDNA purified using the Qiagen PCR purification kit. Labeling reactions with Alexa Fluor 555 or Alexa Fluor 647 fluorescent dyes were performed for 2 h at room temperature. Differentially labeled cDNAs from the two strains to be cohybridized were combined, dried in a Savant SVC100 Speed-Vac (Farmingdale) and stored at -80ºC until hybridization.
|
| |
|
| Channel 2 |
| Source name |
Listeria monocytogenes EGD delta LhrA
|
| Organism |
Listeria monocytogenes |
| Characteristics |
strain: EGD
genotype/variation: delta LhrA
|
| Growth protocol |
Strains were grown to early stationary phase in BHI broth at 37C, defined as OD 1 + 3h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the RNeasy mini or midi kit from Qiagen as described by the manufacturer. Cells grown to early-stationary phase were first treated with RNA protect as instructed by the manufacturer (Qiagen) and subsequently disrupted by sonication on ice (3 X 30 seconds, each round followed by a 30 second pause). RNA was then extracted using the Qiagen RNeasy kit. The integrity of the RNA was confirmed by agarose gel electrophoresis and the concentration and purity was determined on a NanoDrop 2000.
|
| Label |
Cy3 or Cy5
|
| Label protocol |
cDNA from each strain was labeled twice with Cy3 and twice with Cy5 to minimize any bias. cDNA synthesis and labeling of total RNA were performed using the SuperScript Plus indirect cDNA labeling system for DNA microarrays (Invitrogen). 10 µg total RNA was mixed with 5 µg random hexamers and incubated for 10 minutes at 70ºC, with a subsequent chill on ice for at least 5 minutes. Superscript III RT, amino-modified deoxynucleoside triphophates, dithiothreitol, RNaseOUT, and buffer was then added and the reaction mix incubated at 42ºC for 17 hours. RNA was hydrolyzed by the addition of 10 µl 1M NaOH and 10 µl 0.5 M EDTA, followed by incubation at 65ºC for 15 minutes. The mixture was neutralized with 10 µl 1M HCl and cDNA purified using the Qiagen PCR purification kit. Labeling reactions with Alexa Fluor 555 or Alexa Fluor 647 fluorescent dyes were performed for 2 h at room temperature. Differentially labeled cDNAs from the two strains to be cohybridized were combined, dried in a Savant SVC100 Speed-Vac (Farmingdale) and stored at -80ºC until hybridization.
|
| |
|
| |
| Hybridization protocol |
Spotted microarray slides were first incubated for 1 h in a 1% bovine serum albumin-5X SSC-0.1% sodium dodecyl sulfate solution pre-warmed to 42ºC. Subsequently, slides were washed twice in 0.1X SSC and twice in filtered water and then dried. The combined cDNA targets were reconstituted in 55 µl hybridization buffer and denatured at 95ºC for 5 minutes. Targets were applied to microarray slides and overlaid with mSeries LifterSlips (Erie Scientific) followed by overnight hybridization at 42ºC. Slides were then washed 5 minutes in 42ºC pre-warmed 2X SSC plus 0.1% SDS, 5 minutes in 2X SCC, and 2.5 min in 0.2X SSC.
|
| Scan protocol |
After a final wash in filtered water, slides were dried and scanned with a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA).
|
| Data processing |
The median fluorescence intensity data for all probes on the array were analyzed using LIMMA (linear models for microarray analysis). We first normalized the data on each array using print-tip Lowess, followed by log2 conversion of the normalized data. A correlation was determined for the signal from duplicate spots of each probe. Significant differences were determined by calculating a moderated t-statistic which is similar to an ordinary t-statistic except that the standard errors have been shrunk towards a common value using a Bayesian model. P-values were calculated for each gene based on the moderated t-statistics and adjusted with the Benjamini-Hochberg false discovery rate correction for multiple tests. Differences in transcripts levels were considered meaningful only when adjusted P < 0.05 and fold change > 1.5.
|
| |
|
| Submission date |
Jan 24, 2011 |
| Last update date |
Jan 26, 2011 |
| Contact name |
Teresa M Bergholz |
| E-mail(s) |
[email protected]
|
| Organization name |
Cornell University
|
| Department |
Food Science
|
| Lab |
Food Safety Lab
|
| Street address |
405 Stocking Hall
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL5029 |
| Series (1) |
| GSE26818 |
A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes |
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