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Laser Capture Microdissection Service |
The heterogeneous nature of tissue has proven to be the limiting
factor in the amount of biological information that can be generated
from tissue samples. To measure the gene expression profile
with the homogenous cell population, Welgene has used Laser
capture microdissection (LCM) system, PixCell II, to facilitate
the relative research.
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The dissection image
of LCM process (Figure 1) |
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1a. The microscopic examination of the tissue
dissection before LCM capture |
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| Figure 1b.
The microscopic examination of the tissue dissection after LCM
capture |
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| Figure
1c. The target cells captured on the cap |
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Technique
of laser capture microdissection (Figure 2) |
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This robust LCM technology, which was developed at NIH in 1996 and
marketed by Arcturus, has become a universally accepted tool in institutions
worldwide.
LCM uses a nondamaging infrared laser to adhere specific cells to
a thermoplastic film. This ensures integrity of biological molecules
like RNA, DNA, and proteins, which are extracted from the targeted
cells.(Figure 2)
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We have succeeded
to capture single cell by LCM technology¡]Figure 1c ¡^and couple to
downstream analysis. For the microarray experiment, we usually capture
800-2,000 cells. |
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Currently
we provide the cell capturing service by Pixcell II „¥ system
(Figure 3). The service items includes: |
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1. |
Cell capture service:
We suggest frozen dissections for the RNA purification and the
relative downstream process. For the purpose of DNA isolation,
the FFPE or frozen dissections are both suitable. |
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2. |
Cell capture and DNA purification:
Customers are suggested to prepare FFPE or frozen dissections
with a thickness of 7~ 8um. |
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PixCell II„¥ (Figure 3) |
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3. |
Cell capture and RNA purification:
Customers are suggested to prepare FFPE dissections. For the tedious
RNA operation,
LCM sample check list is recommended as a reference.
The workflow of the cell capture and RNA isolation service are shown
in the figure 4.
- Customers should provide the tissue dissections of at least 1000
target cells.
- The first or the last dissection would be chosen for the RNA QC.
After the RNA isolation from the scratched tissues, the RNA is run
on 2100 bioanalzer. By analyzing with highly sensitive capillary electrophoresis
Agilent
RNA 6000 Pico chip, RNA integrity is examined.
- After the RNA QC pass, we¡¦ll make an appointment with customers
to confirm the criteria for the target cell capture. The RNA QC of
the cell captured would be confirmed by bioanalyzer, and cell capture
images are also provided |
Workflow of the cell capture and RNA isolation
service (Figure 4)
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The RNA amount getting from the cells captured is usually insufficient
for microarray analysis. Therefore, we provides RNA linear amplification
to couple these two process. The principle of the reaction is based
on T7 in vitro transcription. The steps includes:
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1. |
The first stranded cDNA synthesis:
The first strand DNA synthesis is conducted by the anchorage
of the oligo-dT-T7 promoter primer to the 3¡¦A-tail of the
mRNA, and the reverse transcription of MMLV RT (Moloney murine
leukemia virus reverse transcriptase). |
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Linear RNA amplification (figure 5) |
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Double stranded cDNA synthesis:
Hydrolysis of the template RNA by RNaseactivity of the MMTV
RT generates the primers for 2nd strand cDNA synthesis. The
5¡¦-end of the resulting cDNA are capped with T7 prometer sequence
and can be served as the promoter for the furthering RNA amplification
reaction.
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3. |
in vitro transcription:
T7 RNA polymerase recognizes the T7 promoter sequence of each cDNA,
and transcribe the downstream cRNA sequences.
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4. |
For the QC of the cRNA
amplification reaction, we measure the amplification efficiency of
the reaction by spectrophotometry. Often the amplification rate should
be over 400 fold. Also the success of cRNA amplifications is examined
by Agilent bioanalyzer 2100 to check the length profile of the cRNA.
The distribution of the amplified cRNA are usually arround 200~1000
nt (as shown in the figure 6)¡C |
| cRNA
QC(Figure 6) |
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