In 2003 I started here as trainee to complete my Bachelor of Applied Science-degree, at the Hanzehogeschool Groningen. During this time I've learned different assays like hormone-assays using RIA (Radio Immuno Assay), avian sexdetermination using PCR and preparation and staining of brain-tissues.
After this, during the VICI-project of Simon Verhulst, I joined the embedded research-group animals@work. Together with this group we could develop different assays, and make them work in our lab. These assays are all connected somehow with telomere-shortning and/or oxidative stress.
* Telomere Length assay using TRF (Terminal Restriction Fragments)
Basically we extract DNA from avian red blood cells or tissue, using agarose blocks to prevent shearing while pipetting. After the DNA is extracted, we cut the DNA in little pieces with restriction enzymes, excluding the telomere-sequence. The DNA is then separated with PFGE (Pulsed Field Gel Electrophoresis), the little pieces run quickly, leaving the long telomeres on the gel. The single strand overhang of the telomere-DNA is then hybridised with a32P-labeled oligo, and made visible with a phosphorscreen. The migration-distance of the telomeres is compared to a 32P-labeled size-ladder, and the data is used for further research.
- Species: Jackdaw, Zebrafinch, Chicken, Oystercatcher, Great Tit
* Telomere Length using qPCR
Besides using the TRF assay, we also use a qPCR-machine to measure telomere-length. In this assay we run samples two times (in triplo), once with a specific primerset which duplicates all telometric regions in the genome and once with a primerset designed on a single copy control gene (like GAPDH). Both Cq values are then calculated and compared. We can approach this with a relative method, but also with a absolute method, where the output is compared to standardcurves of both telomere oligomer and controlgene.
- Species: Zebrafinch, Jackdaw
* Oxidative Stress analysis using OXY and dROM-kit from Diacron
These assays are measured in plasma, and use the Fenton-reaction. With the dROM-kit we measure the level of hydroperoxides, which are derived from damage to lipid and protiens. With the OXY-kit we look at the other side, the antioxidant power of the plasma barrier. Here we colorize the residual hypochlorites. Both assays are colorimetric and are done on a spectrophotometer and platereader.
- Species: Zebrafinch, Jackdaw, Great Tit, Garden Warbler
* Nitric Oxide assay using the Griess reagent system
With this assay we measure both Nitrate (NO3) and Nitrite (NO2) in plasma. To measure both, nitrate needs to be converted to nitrite, catalyzed by cadmium. The amount of nitrite is then assayed using the Griess-reaction, which gives a pink color and this can by quantified colorimetric using a platereader.
- Species: Zebrafinch, Jackdaw, Pigeon
This is also a colorimetric assay. With this assay we make a dilutioncurve with Lutein and compare this with plasma. Both are pipetted in a plate and read with a platereader.
- Species: Chicken, Garden Warbler
These assays are done on a regular base. We also learn students how to do these techniques and I'm developing new assays that fit in our research-questions and possibilities.
|Last modified:||01 May 2015 11.32 a.m.|