|
| Status |
Public on Jan 01, 2011 |
| Title |
10 minutes bile exposure replicate D |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA 10 minutes after bile exposure
|
| Organism |
Lacticaseibacillus rhamnosus |
| Characteristics |
strain: GG
|
| Growth protocol |
Bacterial cells were grown in MRS broth at bioreactor at 37 °C. pH was maintained constant at 6.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
One to three milliliters of bacterial culture were mixed with 2–6 mL of RNAprotect Bacteria reagent (Qiagen) and handled according to the manufacturer’s instructions. The cell pellets were stored at –70 °C for subsequent RNA extraction. Cells were lysed with 10 mg/mL lysozyme (Amresco), 3 mg/mL proteinase K (Sigma-Aldrich), and 100 U of mutanolysin (Sigma-Aldrich) at 37 °C for 30 minutes. The suspension was supplemented with 1 mL of preheated (65 °C) TRIzol reagent (Invitrogen) and vortexed for 3 minutes. After incubation at RT for 5 minutes, the cell lysate was homogenized in a MagNA Lyser instrument (Roche) with <106 µm glass beads (Sigma-Aldrich) for four 30 s cycles at 6000 rpm. Between each cycle, the cells were chilled on ice for 1 minute. Cell debris was removed by centrifugation (12000 × g at 4 °C for 15 min), and the lysate was extracted with 200 µl of chloroform by vortexing for 15 s. After incubation at RT for 3 min, the phases were separated by centrifugation (12000 × g at 4 °C for 15 min). The aqueous phase was mixed with 500 µl of 80% ethanol for total RNA purification with an RNeasy Mini Kit (Qiagen). During RNA purification, DNA was removed using RNase-Free DNase (Qiagen) as described in the manual.
|
| Label |
Cy3
|
| Label protocol |
Five micrograms of RNA were reverse transcribed to cDNA with the SuperScript Indirect cDNA Labeling System (Invitrogen) according to the manufacturer’s protocol except that 6 µg of random primers (Invitrogen, 48190-011) were used instead of anchored oligo(dT)20 primers and random hexamers. The cDNA was fluorescently labeled using Cy3 or Cy5 mono-reactive dyes (Amersham Biosciences) and purified with a column included in the SuperScript Indirect cDNA Labeling System kit.
|
| |
|
| Channel 2 |
| Source name |
Total RNA before bile exposure
|
| Organism |
Lacticaseibacillus rhamnosus |
| Characteristics |
strain: GG
|
| Growth protocol |
Bacterial cells were grown in MRS broth at bioreactor at 37 °C. pH was maintained constant at 6.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
One to three milliliters of bacterial culture were mixed with 2–6 mL of RNAprotect Bacteria reagent (Qiagen) and handled according to the manufacturer’s instructions. The cell pellets were stored at –70 °C for subsequent RNA extraction. Cells were lysed with 10 mg/mL lysozyme (Amresco), 3 mg/mL proteinase K (Sigma-Aldrich), and 100 U of mutanolysin (Sigma-Aldrich) at 37 °C for 30 minutes. The suspension was supplemented with 1 mL of preheated (65 °C) TRIzol reagent (Invitrogen) and vortexed for 3 minutes. After incubation at RT for 5 minutes, the cell lysate was homogenized in a MagNA Lyser instrument (Roche) with <106 µm glass beads (Sigma-Aldrich) for four 30 s cycles at 6000 rpm. Between each cycle, the cells were chilled on ice for 1 minute. Cell debris was removed by centrifugation (12000 × g at 4 °C for 15 min), and the lysate was extracted with 200 µl of chloroform by vortexing for 15 s. After incubation at RT for 3 min, the phases were separated by centrifugation (12000 × g at 4 °C for 15 min). The aqueous phase was mixed with 500 µl of 80% ethanol for total RNA purification with an RNeasy Mini Kit (Qiagen). During RNA purification, DNA was removed using RNase-Free DNase (Qiagen) as described in the manual.
|
| Label |
Cy5
|
| Label protocol |
Five micrograms of RNA were reverse transcribed to cDNA with the SuperScript Indirect cDNA Labeling System (Invitrogen) according to the manufacturer’s protocol except that 6 µg of random primers (Invitrogen, 48190-011) were used instead of anchored oligo(dT)20 primers and random hexamers. The cDNA was fluorescently labeled using Cy3 or Cy5 mono-reactive dyes (Amersham Biosciences) and purified with a column included in the SuperScript Indirect cDNA Labeling System kit.
|
| |
|
| |
| Hybridization protocol |
The labeled cDNA samples were hybridized to microarrays following Agilent’s procedure titled “Two-Color Microarray-Based Gene Expression Analysis.” For each microarray, 825 ng of cyanine 3-labeled cDNA and 825 ng of cyanine 5-labeled cDNA were mixed with 11 µl of 10X GE Blocking Agent and 2.2 µl of 25X Fragmentation Buffer. Volume of this fragmentation mix was brought to 55 µl with nuclease-free water and mix was incubated at 60 °C for exactly 30 minutes. After incubation, fragmentation reaction was stopped by adding 55 µl of 2X Hi-RPM Hybridization Buffer, mixed gently and centrifuged for one minute at RT at 15800 × g. 100 µl of hybridization sample was loaded onto an array and hybridized at 65 °C for 17 hours at 10 rpm with Agilent hybridization oven. Microarrays were washed twice with GE Wash Buffer 1 for one minute at RT and once with prewarmed (37 °C) GE Wash Buffer 2 for one minute.
|
| Scan protocol |
Immediately after washing, microarrays were scanned at 5 µm resolution with a GenePix 4200 AL scanner (Axon Instruments) and stored as TIFF images. The fluorescence intensities were quantified and addressed to genomic ORFs with GenePix Pro 6.0 software (Axon Instruments / Molecular Devices Corp.). Microarray image analysis and feature detection were performed using GenePix Pro 6.0 software with default parameters, and results were further improved manually.
|
| Data processing |
Data analysis was performed using Bioconductor for the R statistical software. Background correction and normalization were done using the limma package, and the statistical significance was assessed using cyberT. The data set contained 12 two-color microarrays, with each condition measured four times, and was analyzed as an entity. The foreground and background median intensity estimates were used, and the data were background corrected using the normexp-function (offset set to 50), normalized within arrays using loess (100 iterations, suspicious spots and probe sequences deleted, matching multiple hits or borders of the coding regions downweighted to zero and intergenic probe sequences downweighted to 0.1) and normalized between arrays using quantile normalization. Expression ratios for genes were obtained by taking the averages of the log2-transformed expression ratios of probes describing the same gene and matching a single genetic coding region locus. The gene expression ratios were calculated for 2798 genes out of the total 2944 (95% coverage) genes in the genome. The statistical significance of the expression ratio of a gene between two conditions was analyzed using a paired t-test method implemented in CyberT (Bayesian prior estimate of within-treatment variance was set to five, and the window size was set to 101). P-values were Bonferroni adjusted by the number of performed t-tests in total (8394). The analysis showed that 248, 133, and 9 genes had significant differences in expression between the zero and 10 min conditions, the zero and 30 min conditions, and zero and 120 min conditions, respectively, when using 0.01 as the threshold for statistical significance and when requiring at least two-fold changes in expression ratio.
|
| |
|
| Submission date |
Jun 23, 2010 |
| Last update date |
Jan 01, 2011 |
| Contact name |
Kati Laakso |
| E-mail(s) |
[email protected]
|
| Organization name |
Valio Ltd
|
| Department |
R&D
|
| Street address |
Meijeritie 4
|
| City |
Helsinki |
| ZIP/Postal code |
00370 |
| Country |
Finland |
| |
|
| Platform ID |
GPL10580 |
| Series (1) |
| GSE22536 |
Bile stress response in probiotic Lactobacillus rhamnosus GG |
|
