|
| Status |
Public on Jun 13, 2011 |
| Title |
qRT-PCR on Staphylococcus aureus strain TB15 in liver |
| Sample type |
RNA |
| |
|
| Source name |
Staphylococcus aureus strain TB15 during mouse liver infection
|
| Organism |
Staphylococcus aureus |
| Characteristics |
sample source: mouse liver infection
strain: TB15
|
| Treatment protocol |
The murine liver model experiment were carried out by following the procedures reported. Strain TB15 was grown overnight in 3 ml TSB and then diluted 1:100 into fresh TSB in 50ml TSB in a 500 ml flask to OD660 = 0.5. The bacterial cells were collected by centrifugation, washed once with 10ml PBS and then suspended in 1.25 ml PBS. In this volume, the bacterial concentration were 2× 108 CFU per 30 ul volume. Six week-old BALB/c mice (10 mice per strain) were anesthetized with Avertin and then inoculated with 30 µl of the cell suspension. After being held upright for 1 minute, the mice were placed into the cage in a supine position. At 72 hr post-infection, the mice were euthanized by CO2 asphyxiation and the livers were harvested. The harvested kidnesy were homogenized and the bacterial numbers were measured by diluting and spreading of the lung homogenate on TSA agar plates. We recovered 106 – 108 CFU from the liver homogenate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ambion mirVana RNA extraction
|
| Label |
Sybr Green
|
| Label protocol |
N/A
|
| |
|
| Hybridization protocol |
RNA samples were reverse transcribed to produce cDNA using SuperScript III (Invitrogen) and then subjected to qRT-PCR using Roche Sybr Green 2x matermix on the Roche 480 Light Cycler in 384-well format. Genes for each sample were represented in duplicates. Reactions were carried out at 10ul final volume. Each reaction contained 10ng of total RNA. PCR activation at 95°C for 10 min was followed by 70 cycles of 15 s at 95°C, 10 s at 55°C and 10 s at 70°C. Sybr green fluorescence was captured in each cycle following 70°C extension.
|
| Scan protocol |
Expression levels were reported in Cp values.
|
| Data processing |
Data was produced in duplicates. The internal controls set was selected and included all genes present in all samples from the same organ with Cp value below 40. The average of the control set for the sample was extracted from the minimum of 2 duplicates. All the data were done in duplicate. We selected the lowest Cp value among two to represent the true Cp value because we had some evaporation problems on some plates that gave unreasonably high Cp values for the number of wells. We did not have designated internal controls on our PCR plates. So we selected all the genes with the Cp values below 40 that present in all samples serve as an internal control set. From our experience Cp values below 40 are more precise and have better reproducibility. We took the average Cp value of the control set Cp(control)for each sample. This Cp(control) is a reflection of how much of the sample total RNA was used in the reaction. Then we normalize (shifted) each Cp value for the sample on that sample Cp(control): Cp- Cp(control).
|
| |
|
| Submission date |
Oct 06, 2010 |
| Last update date |
Jun 13, 2011 |
| Contact name |
John Braisted |
| E-mail(s) |
jbraisted@jcvi.org
|
| Organization name |
J Craig Venter Institute
|
| Department |
PFGRC
|
| Lab |
PFGRC_EXTSW
|
| Street address |
9704 Medical Center Dr
|
| City |
Rockville |
| State/province |
MD |
| ZIP/Postal code |
20850 |
| Country |
USA |
| |
|
| Platform ID |
GPL10963 |
| Series (1) |
| GSE24701 |
Full genome qRT PCR of Staphylococcus aureus strains USA300 and TB15 during infection of 5 organs |
|
