Expression profiling data has proven useful in safety lead optimization and dose-limited toxicity studies, yielding insights sometimes not gleaned from traditional methods. Charles River offers low- and mid-density mouse and rat gene expression testing using pre-validated TaqMan® gene expression assays from Life Technologies. A variety of tissues may be submitted for RNA isolation and quantification. RNA extractions are performed in a high-throughput 96-well format. The quality and quantity of the RNA is assessed by gel electrophoresis and spectrophotometric analysis, respectively. Once RNA is extracted, cDNA is made, testing is performed, and the final data set is analyzed.
Our expression testing is performed on Life Technologies 7900HT or QuantStudio 12K Flex instrumentation. Depending on the number of samples, we can process single-tube samples, 96-well plates, 384-well plates, 384-well microfluidic cards, and 3,072-through-hole OpenArray plates, providing optimized efficiency regardless of sample volume. Utilizing our high-throughput platforms and pre-validated assays, Charles River can perform studies that involve multiple dose groups with samples from several organ systems, including liver, kidney, heart and skeletal muscle. The analysis software has ∆∆Ct calculations for relative RNA quantitation, comparing treated animals with untreated animals or controls.
Calculation by ∆∆ Ct Method
| Sample |
Detector |
Task |
Ct |
Endo Ct Avg |
∆ Ct |
∆∆ Ct |
RQ |
| 1 |
Gene 1 |
Target |
32.70 |
11.20 |
21.49 |
1.21 |
0.43 |
| 1 |
Gene 2 |
Target |
25.27 |
11.20 |
14.08 |
0.05 |
0.97 |
| 1 |
Gene 3 |
Target |
28.70 |
11.20 |
17.55 |
-0.25 |
1.19 |
| 1 |
Gene 4 |
Target |
27.93 |
11.20 |
16.71 |
-0.80 |
1.75 |
| 1 |
GAPDH |
Target |
19.18 |
11.20 |
7.98 |
0.11 |
0.93 |
| 1 |
18sRNA |
Endo Control |
11.20 |
11.20 |
0.00 |
0.00 |
1.00 |
| Control |
Gene 1 |
Target |
31.49 |
11.21 |
20.28 |
- |
- |
| Control |
Gene 2 |
Target |
25.24 |
11.21 |
14.04 |
- |
- |
| Control |
Gene 3 |
Target |
29.00 |
11.21 |
17.79 |
- |
- |
| Control |
Gene 4 |
Target |
28.72 |
11.21 |
17.51 |
- |
- |
| Control |
GAPDH |
Target |
19.08 |
11.21 |
7.87 |
- |
- |
| Control |
18sRNA |
Endo Control |
11.21 |
11.21 |
0.00 |
- |
- |
Samples are tested in duplicate along with the endogenous control (18sRNA). ∆Ct values are calculated from the average target Ct values and the average endogenous Ct values. ∆∆Ct values are derived by using a known untreated or control for respective genes. Final relative quantitation (RQ) values are derived from ∆∆Ct values using the formula 2^-∆∆Ct. The RQ values represent the up or down regulation of gene expression in the sample relative to the control.
Amplification Figure
The data generated from these tests provides clients with early indications of toxicity and efficacy prior to moving on to more intensive, and costly, studies in other species. Custom assays on other tissues or species are also possible, and each custom assay is developed in-house and is rigorously optimized and validated prior to use. Charles River has considerable scientific and technical expertise in the development of assays for gene expression studies or target gene detection.
Expression testing can also be used to confirm data for individual or multiple genes obtained from large-scale research studies or to quantify the effect of RNA-mediated gene knockdown. Quantitative reverse transcription PCR (QRT-PCR) measures the steady-state level of mRNA expression from a transgene or knockout construct. Expression profiling data has proven useful in screening various compounds for toxicity or pharmaceutical value.
For more information, please contact us at 1-877-CRIVER-1 or askcharlesriver@crl.com.