|
| Status |
Public on Feb 25, 2010 |
| Title |
Turkey 3-3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from Listeria monocytogenes F2365 labeled with cy5.
|
| Organism |
Listeria monocytogenes |
| Characteristics |
media: agar
|
| Biomaterial provider |
Chinling Wang at Mississippi State University
|
| Treatment protocol |
hybridization:
image_acquisition:
labeling:
reverse_transcription:
spin:
wash:
|
| Growth protocol |
About 2.3 x 10^8 CFU/ml was inoculated aseptically in BHI agar plate and incubated at 15 °C for 5 days in a BOD10 refrigerated incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA Protect® (Qiagen, Valencia, CA) was added in bacterial culture at a ratio of 2:1 before collecting cells. Total RNA was extracted from the bacterial pellet, and purified using Trizol® (Invitrogen, Carlsbad, CA) and the RNeasy® Mini Kit (Qiagen), respectively.
|
| Label |
Cy5
|
| Label protocol |
To synthesize cDNA, 2.5 ug of total RNA was reversely transcribed. cDNA was synthesized using 2 ul of Smart® reverse transcriptase (Clontech, Palo Alto, CA) and 1 ul of random hexamers in the presence of 0.1 M dithiothreitol (DTT) [Invitrogen], 25 mM dNTP with 3:2 ratio of aminoallyl (aa)-dUTP and dTTP (Ambion, Austin, TX). After cDNA synthesis, the cDNA was purified and unincorpotated aa-dUTP was removed using a MinElute® PCR purification kit (Qiagen). The purified cDNA resuspended in 0.1 M sodium carbonate buffer (pH 9.3) was labelled with cy5.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from Listeria monocytogenes F2365 labeled with cy3.
|
| Organism |
Listeria monocytogenes |
| Characteristics |
nutrients: turkey matrix
|
| Biomaterial provider |
Chinling Wang at Mississippi State University
|
| Treatment protocol |
hybridization:
image_acquisition:
labeling:
reverse_transcription:
spin:
wash:
|
| Growth protocol |
About 2.3 x 10^8 CFU/ml was inoculated aseptically on a turkey meat slice in a sterile stomacher bag and incubated at 15 °C for 5 days in a BOD10 refrigerated incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA Protect® (Qiagen, Valencia, CA) was added in bacterial culture at a ratio of 2:1 before collecting cells. Total RNA was extracted from the bacterial pellet, and purified using Trizol® (Invitrogen, Carlsbad, CA) and the RNeasy® Mini Kit (Qiagen), respectively.
|
| Label |
Cy3
|
| Label protocol |
To synthesize cDNA, 2.5 ug of total RNA was reversely transcribed. cDNA was synthesized using 2 ul of Smart® reverse transcriptase (Clontech, Palo Alto, CA) and 1 ul of random hexamers in the presence of 0.1 M dithiothreitol (DTT) [Invitrogen], 25 mM dNTP with 3:2 ratio of aminoallyl (aa)-dUTP and dTTP (Ambion, Austin, TX). After cDNA synthesis, the cDNA was purified and unincorpotated aa-dUTP was removed using a MinElute® PCR purification kit (Qiagen). The purified cDNA resuspended in 0.1 M sodium carbonate buffer (pH 9.3) was labelled with cy3.
|
| |
|
| |
| Hybridization protocol |
About 5.8 ug of each labelled cDNA were hybridized, resuspended in 50 ml hybridization buffer (40% formamide, 5X sodium chloride/sodium citrate buffer, 0.1% SDS, and 0.6 ug/ul salmon sperm DNA) and loaded on a microarray slide. The slide was incubated for 16h at 42 °C, and then washed with low, medium, and high stringency buffer containing 0.1 mM DTT.
|
| Scan protocol |
The hybridized slides were scanned at 10 um resolutions within the range of photomultiplier tube between 650 and 750 on two channels (cy3 and cy5) using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA).
|
| Description |
Data were obtained from three biological and four technical replicates (n = 12). For each biological replicate, flip dye analysis was performed. The expression level of genes obtained from the mean of each gene on the twelve slides was considered significantly different over 1.5 fold changes. The difference was presented at a significant level (P < 0.05).
|
| Data processing |
The signal intensity of spots on the slides was adjusted and quantified by background subtraction using Spotfinder software. Data LOWESS normalization was performed using Ginkgo software. The data from the normalized ratio of query to reference signal for each spot were transformed using a log2 scale.
|
| |
|
| Submission date |
Feb 10, 2010 |
| Last update date |
Feb 24, 2010 |
| Contact name |
Chinling Wang |
| E-mail(s) |
wang@cvm.msstate.edu
|
| Phone |
662-325-1683
|
| Fax |
662-325-1031
|
| Organization name |
Missippi State University
|
| Department |
Basic Sciences
|
| Lab |
Proteomics
|
| Street address |
College of Veterinary Medicine P.O. box 6100
|
| City |
Mississippi State |
| State/province |
MS |
| ZIP/Postal code |
39762 |
| Country |
USA |
| |
|
| Platform ID |
GPL5854 |
| Series (1) |
| GSE20274 |
Transcriptome profiling of Listeria monocytogenes during growth on a RTE-meat matrix |
|
