Automated in situ
hybridization of microRNA on the DISCOVERY ULTRA
Introduction
MicroRNAs
(miRNA) are small non-coding RNA that are typically 18-22 base pairs
in length. Due to observations of their ability to regulate mRNA
transcripts that can result in translational repression and gene
silencing, they have been a topic of high interest for researchers.
Ventana Medical Systems, Inc. has developed a robust and reproducible
assay on the DISCOVERY ULTRA instruments for the in
situ detection of miRNA by using Exiqon LNA probes.
Materials and Methods
UltraMap anti-Mouse AP (Ventana p/n 760-4312)
ChromoMap Blue Kit (Ventana p/n 760-161)
250 test Enzyme 1 User Fillable Dispenser (Ventana p/n 771-721)
250 test Antibody 1 User Fillable Dispenser (Ventana p/n 770-001)
250 test Probe 1, 2, 3 User Fillable Dispensers (Ventana p/n 960-761,
960-762, 960-763)
Mouse anti-DIG (Roche Applied Science p/n 11333062910)
DISCOVERY Antibody Diluent (Ventana p/n 760-108)
Red Counterstain II (Ventana p/n 780-2218)
microRNA ISH Optimization Kit 8 FFPE (Exiqon p/n 90008)
hsa-miR-126 (Exiqon p/n 88067-15)
Optional reagents needed for signal amplification:
OmniMap anti-Mouse HRP (Ventana p/n 760-4310)
DISCOVERY Amplification HQ kit (Ventana p/n 760-052)
DISCOVERY anti-HQ AP Multimer (Ventana p/n 760-4521)
Each of the following reagents was diluted and placed in a
respective user-fillable dispenser (enzyme, antibody, and probes) for
complete automation of the experiment on the DISCOVERY ULTRA
instrument. The proteinase K provided in the Exiqon optimization kit
was reconstituted and diluted to 15ug/mL with a 5mM Tris buffer pH 7.3
and 1mM EDTA. The Mouse anti-DIG was diluted to 2ug/mL in DISCOVERY
Antibody Diluent. A 7.5nM solution was made of each of the scramble,
miR-126 and miR-205 LNA probes with the hybridization buffer provided
in the optimization kit.
The following selections were made on the “RUO DISCOVERY
Multimer Amp Plus” procedure provided with the DISCOVERY ULTRA Software:
Button Selection
Details
Deparaffinization
Three
cycles of 4 minutes at 65°c
Pretreatment
-Enzyme
-Use RB
for Enzyme
Incubate
the proteinase K at 37°c. Depending on the tissue, incubate the enzyme
from 4-20 minutes
ISH
-1 st Probe
Denature
at 80°c for 8 minutes. Hybridize the probe at 30°c below the Tm
1 st Stringency Wash
2.0x SSC
at hybe temperature for 4 minutes
2 nd Stringency Wash
2.0x SSC
at hybe temperature for 4 minutes
Antibody
Incubate
the anti-DIG at 37°c for 20 minutes
DISCOVERY Detection
-Disable Inhibitor CM
-AP
detection
Incubate
UltraMap anti-Mouse AP for 16 minutes
DISCOVERY Amplification ***
-Discovery
Amplification HQ
Incubate OmniMap anti-Mouse
for 16 minutes
Incubate Amp HQ and Amp
H2O2
for 24 minutes
Incubate
anti-HQ AP for 16 minutes
Blue
Incubate
substrate for 44 minutes
Counterstain
-Use DW
for Counterstain
Incubate
Red Counterstain II for 8 minutes
***If amplification is to be used
Results
Conclusion
Due to the flexible nature of the DISCOVERY ULTRA platform, it
is an ideal instrument for research and optimization of in situ
protocols, particularly miRNA. The ability to fully automate the whole
in situ process makes the DISCOVERY ULTRA platform a valuable research
tool. Furthermore, the flexibility of the instrument allows for the
intensity of signal to be customizable, which can be increased with the
incorporation of Amp-HQ without increasing any background.
Reference
Stine Jorgensen, Adam Baker, Soren Moller, Boye Schnack
Nielsen. Robust one-day in situ
hybridization protocol for detection of microRNAs in paraffin samples
using LNA probes. 2010. Methods. 52:375-381.
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