This practice themetest contains questions from mainly the expert, theory of the practicals, tutor lectures and practical classes. The answers are written on the last 2 pages.
Practice test – Theme test BLM
Course 10
*In this practice test is mainly focussed on the theory derived from the course introduction, theory of the
practicals, tutor and expert lectures. Theory from OMICS and the workshops are not included.
Melanomas (9 pnt.)
During the practicals, we have investigated the effect of ALCAM-ALCAM interactions on gene
expression and MMP2 activation.
A) What is ALCAM? And why is this molecule interesting to us in melanoma research? (Include
something about ‘Clark level’ in your answer) (3 pnt.)
B) Draw globally the ALCAM molecule and name the different components of this molecule. (2 pnt.)
C) In the practicals, we have also worked with 2 mutants of the ALCAM molecule: -dN and soluble
ALCAM. Explain in detail why the ALCAM-ALCAM network formation is disrupted in both mutants. (4
pnt.)
MMP2 activation (13 pnt.)
ALCAM expression and MMP2 activation are correlated to each other and are both expressed at the
invasive front of a melanoma. MMP2 = matrix metalloproteinase 2.
A) Explain, with your knowledge about the function of MMP2, why it is thought by researchers that
MMP2 can facilitate tumour metastasis (hypothesis). (4 pnt.)
B) How does the activation of MMP2 take place? Explain this on molecular level. (4 pnt.)
In the practicals, the MMP2 activation is investigated by collecting medium from the 3D cultures and
putting these samples on a zymography gel.
C) Name 1 other technique that can be used to investigate the invasiveness of a cell line and explain
how this technique works. (2 pnt.)
D) A sample buffer needs to be added to the samples for the zymography gel, before they can be
loaded onto the gel. In the sample buffer are the components: glycerol, SDS and bromophenol blue.
Explain for each component what its function is. (2 pnt.)
E) Why is there normally added DTT/BME to the sample buffer, but is it wrong to add to the samples
of the zymography? (1 pnt.)
Gene expression (10 pnt.)
We started this block with the cell culture pellets from the technicians. The pellets that we got were
the parental, -dN and -s cell lines in the 60% and 200% confluency. A few experimental steps were
needed to go from pellet to cDNA which was needed for the qPCR.
A) Name a disadvantage of the qPCR technique and name another technique that can be used to
investigate gene expression of a certain gene in a sample. (1 pnt.)
, B) The conversion from mRNA to cDNA was needed for the qPCR and is performed by cDNA
synthesis. Multiple different primers could be used for the reversed transcription. Name 1 of the 3
possible primers and name a disadvantage or advantage of this primer. (2 pnt.)
C) There are different detection mechanisms possible to detect the fluorescence in the qPCR. WE
used SYBR-green in the lab. Explain shortly how the fluorescence detection works using SYBR-green
in the qPCR. (1 pnt.)
D) The following data is derived from a performed qPCR:
Calculate if there is up-/down-regulation of the GOI in the treated BLM cells by using the ddCq
method (3 pnt.) and the Pfaffl method (3 pnt.). The efficiency of the primers is in this case 100%.
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