Molecular characterisation of SGCE-associated myoclonus-dystonia ...

Patients, material and methods 28
Synergy HT plate reader
BIOTEK
Thermocycler - LightCycler
RocheDiagnostics
- Mastercycler Eppendorf
- 7300 Real-Time PCR System Applied Biosystems
Thermoseparation Products chromejet integrator Anachem
Upstream and downstream electrode
ESA Analytical
2.2.7 Software
geNorm program
Image processing program
RQ Study Application
VBA applet10
TotalLab
7300 SDS Software
2.3 Methods
This methodical description is especially focussed on techniques which were established in
the neurogenetics laboratory of Professor Klein as a part of the present thesis.
2.3.1 Extraction of nucleic acids
During the experimental phase of this thesis DNA was gained from human blood and
fibroblast cell cultures. RNA was only extracted from blood samples.
2.3.1.1 DNA extraction from whole blood
Genomic DNA was prepared from leucocytes of peripheral blood by means of a salting out
method (Miller et al., 1988). Cells are lysed by adding of a hypertonic solution. Proteins are
digested by proteinase K and the DNA is isolated by precipitation.
By means of this method between 100 and 1000μg genomic DNA can be retained.
2.3.1.2 DNA extraction from fibroblasts
For DNA extraction from fibroblast the Nucleon I kit was used. Cell pellets were processed
according to manufacture’s protocol.
2.3.1.3 RNA extraction from whole blood
Blood for RNA extraction from patients was collected in Pax gene tubes and extracted with
the respective Pax gene blood kit.