Using Multiplex Ligation-Dependent Probe Amplification to
Determine the Expression of Androgen Receptor Variants
Introduction
The androgen receptor is a nuclear receptor unique to
prostate cells that activate upon binding to androgenic
hormones such as testosterone. The transcription of
the androgen receptor gene results in a diverse group
of splice variants known as androgen receptor splice
variants. Abnormally high expression of specific
androgen receptor (AR) splice variants in prostate
cells has been linked to the development of higher
grade castration resistant prostate cancer. As a result,
measuring the expression of these AR splice variants
is often used for the diagnosis and prognosis of
prostate cancer.
Objectives
This experiment seeks to use the procedure of
Multiplex Ligation-Dependent Probe Amplification
(MLPA) to maximize the efficiency and effectiveness
of non-invasive methods for detecting prostate
cancers in patients. Specifically, the goals are to:
• Design custom oligonucleotide probes which bind
to the specific exon-exon junctions unique to each
AR splice variant.
• Utilize gel electrophoresis and capillary
electrophoresis to analyze post-PCR products in
order to determine expression levels of each AR
splice variant.
• Compare the experimentally determined expression
level of AR splice variants in prostate cell line cDNA
to manufactured AR splice variant plasmid.
• Reconstruct the experiment to test the viability of
direct-binding custom oligonucleotide probes to
mRNA rather than cDNA
Probe Design
Oligonucleotides Probe Design:
• Probes must be constructed within the parameters
provided by MRC-Holland, the company providing
the MLPA kits.
• Probes were made to bind to exon-exon junctions
unique to each AR splice variant. For instance, a
probe was constructed to bind to the exon 7 to exon
8 junction, a junction unique to the full length AR, in
order to detect the presence of full length AR.
• Similarly, the exon 3 to cryptic exon 3 junction is
used to detect AR-V7, the exon 3 to cryptic exon 5
junction to detect AR-V9, and the exon 4 to exon 8
junction to detect AR-V12. The exon 1 to exon 2
junction exists in all AR splice variants and is used
as a control.
POSTER TEMPLATE BY:
www.PosterPresentations.com
Ruoheng Zeng, Yezi Zhu, Jun Luo, Changxue Lu
Brady Urological Institute, Johns Hopkins Medical Institute
Methods
mRNA Preparation:
1. Extract mRNA from test group cell lines: LNCaP,
LNCaP95, CWR22Rv1, HeLa, DU145, and PC3. The
former three serves as AR positive test group, while
the latter three serve as the AR negative test group.
Control for density between test groups through
dilutions.
2. Perform reverse transcription on mRNA to make
cDNA.
Multiplex Ligation-Dependent Probe Amplification
1. Oligonucleotide probes, consisting of two fragment
pairs, are hybridized with the target nucleic acid
segments in the samples.
2. Pairs of successfully hybridized fragments are
ligated together with enzyme solution.
3. Successfully ligated fragment are amplified through
PCR, and are collected as final MLPA products.
Data Analysis
1. Final MLPA products are analyzed using gel
electrophoresis and capillary electrophoresis.
2. Gel electrophoresis confirms the presence of
successfully ligated and amplified products, but
doesn’t allow for discrimination between the
expression of different AR variants.
3. Capillary electrophoresis is done on the same MLPA
products to quantify the expression levels of each
AR variant in each sample relative to a control.
Follow up experiment:
Cell line cDNA is replaced with raw mRNA, testing the
viability of the MLPA probes direct binding. Enzyme
solutions used in the MLPA, as well as incubation
times, are modified to allow direct binding of mRNA.
Gel Electrophoresis Results
Capillary Electrophoresis Results
Graph 1. Expression of CWR22Rv1
Graph 2. Expression of LNCaP95
Graph 3. Expression of LNCaP
Graph 4. Expression of AR splice variants in
LNCaP95 and CWR22Rv1 normalized to LNCaP
Conclusion
• Multiplex Ligation-dependent Probe Amplification is
effective in determining the differentiated
expression levels of the AR splice variants.
• MLPA data for cDNA from prostate cell lines with
known expression levels correlate well with
expected expression levels. All the AR positive cell
lines (LNCaP, LNCaP95, and CWR22Rv1) show high
levels of expression for full length AR and
significant expression of AR-V9 and AR-V12
• Additionally, as compared to LNCap, a cell line
known for its lack of AR-V7 expression, AR-V7
expression LNCaP95 and CWR22Rv1 expression
for AR-V7.
• MLPA data for probes directed-bound to mRNA
were less consistent across the board. From
capillary electrophoresis analysis, there were clear
peaks indicating the presence of the expected
junctions, but there were also many non-specific
peaks likely due to error.
• The presence of background noise is likely caused
by non-specific bindings in the probes during the
hybridization process. This was especially likely for
mRNA binding, due to the flimsy compatibility of
the necessary ligase with the rest of the MLPA kit
Future Direction
• The future of this study will largely focus on
increasing the precision and accuracy of direct-
binding oligonucleotide probes to mRNA rather
than cDNA.
• There are many advantages to direct-binding to
mRNA, the main ones being a result of bypassing
reverse transcription, which leads to increases in
efficiency and decreases in cost of the experiment.
• The future of MLPA lies in its efficiency and
simplicity as compared to alternative methods of
determining gene expression.
References
Lu CX, Luo J. Decoding the androgen receptor
splice variants. Translational andrology and
urology. 2013. [PubMed: 25356377]
MRC-Holland MLPA. (n.d.). Retrieved February 19,
2019, from MRC-Holland website:
https://www.mlpa.com/
Sharp, A., & Coleman, I. (n.d.). Androgen receptor
splice variant-7 expression emerges with
castration resistance in prostate cancer. Journal of
Clinical Investigation. https://doi.org/
10.1172/JCI122819
Acknowledgement
This work was made possible by the primary
investigator Dr. Jun Luo and post-doc fellow Dr. Yezi
Zhu. Additionally, it is supported by the Brady
Urological Institute at Johns Hopkins Hospital.