08_RTS_rz
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10:17 Uhr
Seite 24
pIVEX Plasmid
GFP
b-Gal
PCR Product
CAT
GFP
b-Gal
CAT
The quality of three different proteins expressed from PCRgenerated templates was analyzed in further detail. In vitro
expression of pIVEX plasmids coding for GFP (28 kDa),
β-galactosidase (β-Gal, 120 kDa), and chloramphenicol
acetyltransferase (CAT, 26 kDa) was compared to the expressions from PCR-generated templates using radioactive labeling with L-[35S] methionine. No DNA templatedependent difference was evident (Figure 3). Even a protein with a size of 120 kDa was sucessfully expressed from
linear DNA.
References
1. Watzele et al., Biochemica 3 (2001): 27.
Figure 3: Autoradiography of in vitro synthesized
proteins: Green fluorescent protein (GFP),
β-galactosidase (β
β-Gal) and chloramphenicol acetyl
transferase (CAT) were expressed in 25-µl RTS 100
reactions in the presence of 1 µM L-[35S] methionine.
After SDS polyacrylamide gel electrophoresis, the dried
gel was exposed for 3 hours
RTS
Kit resulted in more than 200 µg/ml of active GFP.
Compared to conventional two-step PCR expression
methods using long outer primers, the protein yields
were almost doubled when using the PCR templates
generated by the RTS E. coli Linear Template Generation
Set, His-tag (data not shown).
Product
Pack Size
Cat. No.
Rapid Translation System
RTS E. coli Linear Template
Generation Set, His-tag
Rapid Translation System
RTS 100 E. coli HY Kit
96 reactions
(50 µl each)
3 186 237
24 reactions
(50 µl each)
96 reactions
(50 µl each)
2 x 10 µg
3 186 148
3 186 156
100 units
1 732 641
Rapid Translation System
RTS pIVEX His-tag,
2nd Generation Vector Set
Expand High Fidelity PCR
System
3 269 019
FastStart Taq DNA Polymerase Is Ideally Suited for
RT-PCR Using Laser Captured Microdissected Material
Andy Green* and Ian White
MRC Molecular Endocrinology Group, Department of Obstetrics and Gynaecology
University of Leicester, Leicester, UK
*Corresponding author: [email protected]
Introduction
Laser capture microdissection (LCM) is a new
technique allowing the isolation of small
numbers of cells or cell subpopulations from
fixed tissue sections [1]. Downstream applications can
include gene expression studies using the sensitive technique, reverse transcription polymerase chain reaction
Andy Green
24
BIOCHEMICA · NO. 2 · 2002
(RT-PCR). However, when using small quantities of starting material, it is important to obtain high specificity and
sensitivity during the amplification of genes. We have
therefore undertaken a comparative study of commercially available reverse transcriptases and hot start Taq DNA
polymerases to identify the optimal combination for quantification of gene expression in a small quantity of cells
isolated by LCM.
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Seite 25
Material and Methods
GAPDH was amplified in a total volume of 20 µl using five
different hot start Taq DNA polymerases (supplier 1, 2, 3,
4, and FastStart Taq DNA Polymerase) using a thermal
cycler. Each reaction contained the PCR buffer supplied by
the respective manufacturer, 0.2 µM of each primer and
200 µM of dNTP. FastStart Taq DNA Polymerase (0.8 U)
was used with a final concentration of 2 mM MgCl2. The
PCR began with a 4-minute denaturation/activation step
at 95 °C. PCR cycles began with 95 °C for 30 seconds, followed by an annealing temperature of 60 °C and concluded with a 45-second elongation step at 72 °C. This was
repeated for a total of 40 PCR cycles with a final extension
temperature of 72 °C for 7 minutes. Similar cycling conditions were used for the other hot start Taq DNA polymerases following the manufacturers’ instructions. The
resulting 300-bp PCR products were electrophoresed in
parallel through a 2 % agarose gel using a SYBR green
stain, then visualised under UV light.
Supplier 4
FastStart
Supplier 3
Supplier 2
Supplier 1
Supplier 4
FastStart
Supplier 4
FastStart
Supplier 3
Supplier 2
300 bp
Supplier A
Supplier B
Supplier C
Reverse Transcriptase
Figure 1: Comparison of reverse transcriptase and hot start Taq DNA
polymerase efficiency in RT-PCR of GAPDH over 40 cycles of mouse uterine
luminal epithelium cells isolated by laser capture microdissection
by FastStart Taq DNA Polymerase was generally as good
or better compared to hot start Taq DNA polymerases
from other suppliers. However, non-specific amplification
was detected in the majority of PCR from other hot start
Taq DNA polymerase suppliers, whereas it was minimal
with the use of FastStart Taq DNA Polymerase. FastStart
Taq DNA Polymerase was therefore chosen for optimal
PCR amplification of RNA extracted from LCM material.
MOLECULAR BIOLOGY
cDNA was synthesised using random hexamers and
three different reverse transcriptases (supplier A, B, and
C) in a total volume of 20 µl according to the manufacturers’ instructions. The expression of glyceraldehyde6-phosphate dehydrogenase (GAPDH) was determined
using PCR by amplifying 1 µl of cDNA with the primer
sequences 5’-ACCCAGAAGACTGTGGATGG-3’ and
5’-GAGACAACCTGGTCCTCAG-3’.
Supplier 1
Separate populations of mouse luminal epithelial cells
were procured from 8-µm frozen sections of uterus fixed
in 70 % ethanol using the PixCell II Laser Capture
Microdissection System (Arcturus Engineering). An
average of 100 laser shots (15 µm) per sample were
used, yielding approximately 100-200 cells. Total RNA
was extracted from each sample using a commercially
available kit, then divided equally into three.
Hot Start Taq DNA Polymerase
Supplier 3
10:17 Uhr
Supplier 2
20.03.2002
Supplier 1
08_RTS_rz
FastStart Taq DNA Polymerase has also successfully
amplified targets of a lower abundance than GAPDH,
such as estrogen receptor α and β in LCM material.
Summary
We have successfully used FastStart Taq DNA Polymerase
to amplify gene expression targets from limited cell samples isolated by laser capture microdissection. This product provides superior sensitivity and less non-specific
amplification compared with other hot start Taq DNA
polymerases.
References
1. Emmert-Buck M.R. et al. (1996), Science 274: 998-1001.
Results and Discussion
To compare the efficiency of several reverse transcriptases
and hot start Taq DNA polymerases, total RNA was
extracted from luminal epithelial LCM samples of a mouse
uterus. cDNA synthesis was conducted using three different reverse transcriptases, and GAPDH was amplified
using PCR in parallel with five different hot start Taq DNA
polymerases, including FastStart Taq DNA polymerase.
GAPDH cDNA was amplified in each case but with a varying degree of efficiency and specificity when comparing
both reverse transcriptases and hot start Taq DNA polymerases (Figure 1). The degree of amplification of GAPDH
WWW.ROCHE-APPLIED-SCIENCE.COM
Product
Pack Size
Cat. No.
FastStart Taq
DNA Polymerase
50 units
for 25 PCRs
100 units
for 50 PCRs
2 x 250 units
for 250 PCRs
4 x 250 units
for 500 PCRs
10 x 250 units
for 1,250 PCRs
20 x 250 units
for 2,500 PCRs
2 158 264
2 032 902
2 032 929
2 032 937
2 032 945
2 032 953
BIOCHEMICA · NO. 2 · 2002
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