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Molecular Diagnostics and Genetics |
1
Department of Pathology, University of California, Irvine, CA 92612.
2
Second Department of Surgery, Yokohama City University,
School of Medicine, Yokohama 236, Japan.
3
Hitachi Chemical Research Center, 1003 Health Sciences
Road West, Irvine, CA 92612.
a Author for correspondence. Fax 949-725-2727; e-mail mmitsuha{at}uci.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that mRNA is captured successfully by an oligo(dT)-immobilized polystyrene (PS) microplate [GenePlate®, Hitachi Chemical Co., and Advanced Gene Computing Technologies (AGCT)] (3)(4)(5); single- and double-stranded cDNA synthesis then is performed directly on the plate (6)(7). Once double-stranded cDNA is formed on the plate, sense-stranded cDNA can be removed and used as a template for multiple PCR experiments (7). Unfortunately, PCR cannot be performed in this PS GenePlate because of its heat instability. Although heat-stable polypropylene (PP) tubes and microplates are primary vessels for PCR, it is difficult to immobilize oligonucleotides on PP because of its extremely stable surface characteristics. Here, using newly developed oligo(dT)-immobilized PP microplates, we report direct RT-PCR from crude cell lysates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell culture
K562 cells were grown in RPMI-1640 containing 100 mL/L fetal
bovine serum, 500 000 units/L penicillin, and 500 mg/L streptomycin
and subcultured twice a week at a ratio of ~1:10. Cell viability was
assessed by the exclusion of trypan blue and was always >95%.
preparation of cell lysate and total rna
Cells were washed with phosphate-buffered saline twice and
suspended in lysis buffer (10 mmol/L Tris, pH 7.6, 1 mmol/L EDTA, 1 g/L
NP-40, and 20 mmol/L vanadyl ribonucleoside complex) on ice for 5 min
to release cytosolic mRNA as described previously (4).
Samples were then centrifuged at 15 000g at 4 °C for 5
min, and supernatant solutions were applied to the GenePlate-PP for
hybridization. Total RNA was prepared by an automated instrument
(MagExtractor MFX-2000, Toyobo). In brief, cell pellets were suspended
in kit-supplied chaotropic agents and placed in the MagExtractor, where
RNA was absorbed to the surface of magnetizable silica particles,
followed by magnetic separation. RNA was automatically eluted in 40
µL of kit-supplied low-salt buffer and was stored at -80 °C until
use. The final RNA was analyzed by agarose gel electrophoresis to
confirm the presence of 18S and 28S rRNA bands.
quantification of hybridization
After mRNA hybridization on the GenePlate-PP, each well was washed
three times with wash buffer (10 mmol/L Tris, pH 7.6, 300 mmol/L NaCl,
10 mmol/L Tween 20). Fifty microliters of Yoyo-1 was diluted in
Tris-EDTA (10 mmol/L Tris, pH 8.0, 1 mmol/L EDTA) in a final dilution
of 1:1000 and applied to the GenePlate-PP. The fluorescence was
determined by a CytoFluor 2300 (Millipore), with excitation and
emission wavelengths of 485 nm (bandwidth, 20 nm) and 530 nm
(bandwidth, 25 nm), respectively, as described previously
(4)(5). To analyze the reversibility of hybridization, mRNA
was removed by adding warm or hot water, and Yoyo-1 fluorescence was
determined. Moreover, to quantify the capacity of the GenePlate-PP for
hybridized mRNA, the dissociated mRNA was applied to fresh GenePlate-PP
for a second hybridization followed by quantification of cDNA
synthesis, as described below.
quantification of first- and second-strand cDNASYNTHESIS
The amount of cDNA synthesis was quantified according to the
protocol published by Tominaga et al. (6) with minor
modifications. In brief, mRNA hybridized to GenePlate-PP was
resuspended in 50 µL of cDNA synthesis buffer (50 mmol/L Tris, pH
8.3, containing 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L
dithiothreitol, 250 µmol/L each of dATP, dGTP, and dCTP, 25 µmol/L
biotin-dUTP, and 400 U of MMLV reverse transcriptase), and the plates
were incubated at 37 °C for 1 h. After each well was washed
three times with wash buffer (10 mmol/L Tris, pH 7.6, containing 300
mmol/L NaCl and 1.0 mL/L Tween 20), 50 µL of wash buffer containing a
1:1000 dilution of streptavidin-alkaline phosphatase conjugate was
added, and the microplate was incubated at room temperature for 30 min.
After each well was washed three times with wash buffer, 50 µL of
substrate (AttoPhos, 1x concentration) was added, and the plate was
incubated at room temperature for 20 min. The reaction was terminated
by addition of an equal volume of 100 mmol/L EDTA, and the fluorescence
was determined by the CytoFluor 2300 (Millipore), with excitation and
emission wavelengths of 485 nm (bandwidth, 20 nm) and 560 nm
(bandwidth, 25 nm), respectively.
Second-strand cDNA synthesis was also quantified as follows: first-strand cDNA was synthesized according to the protocol described above with use of 250 µmol/L unlabeled dUTP instead of biotin-dUTP. The buffer was replaced with 50 µL of second-strand cDNA synthesis buffer (25 mmol/L Tris, pH 7.5, 100 mmol/L KCl, 5 mmol/L MgCl2, 10 mmol/L (NH4)2SO4, 0.15 mmol/L ß-NAD, 250 µmol/L each of dGTP, dCTP, dTTP, and biotin-dATP or dATP dGTP, dCTP, and biotin-dTTP, 1.2 mmol/L dithiothreitol, 65 kU/L DNA ligase, 250 kU/L DNA polymerase, and 13 kU/L RNase H, and the plate was incubated at 16 °C overnight. The amount of incorporation of biotin was quantified by AttoPhos as described above.
quality of synthesized cDNA
Second-strand cDNA with the biotin-dATP incorporated as described
above was removed from the GenePlate-PP in boiling water and treated
with phenol–chloroform once, followed by ethanol precipitation. The
pellets were suspended in diethylpyrocarbonate (DEPC) water and
separated by 0.8% agarose electrophoresis. After electrophoresis was
completed, gels were placed on supported nitrocellulose membranes
(Optitran) presoaked with 20x standard saline citrate (3 mol/L NaCl,
0.3 mol/L sodium citrate), and cDNA was transferred from gels to
membranes with positive pressure (Posiblot, Stratagene) at 60 mmHg for
1 h. Membranes were exposed to ultraviolet light (Stratalinker,
Stratagene) at 120 mJ and incubated with Genius buffer 1 (Boehringer
Mannheim) containing 3 mL/L Tween 20 for 1 min at room temperature,
followed by Genius buffer 2 for 45 min. The buffer was replaced with
fresh Genius 2 containing 3 mL/L Tween 20 and a 1:10 000 dilution of
streptavidin-alkaline phosphatase conjugate, and the membranes were
incubated for another 30 min at room temperature. The membranes were
washed twice with Genius buffer 2 for 15 min, equilibrated in Genius
buffer 3 for 2 min, and reacted with 1x LumiPhos. Membranes were
placed in plastic bags and exposed to x-ray films.
primer design and synthesis
Primers for rabbit globin mRNA (sense, 5'-cgtggagaggatgttcttgg-3';
antisense, 5'-aacgatatttggaggtcagcac-3') and bcr-abl (sense,
5'-gaccaactcgtgtgtgaaactcca-3'; antisense,
5'-aaagtcagatgctactggccgct-3') were designed by HYBsimulator software
(AGCT) using hybridization simulation against the GenBank primate
database (8)(9). Primers for G3PDH were purchased from
Clontech. In the case of bcr-abl, the sense primer was
located at bcr exon 2, and the antisense primer was located
at abl exon 2. Primers were synthesized in a DNA synthesizer
(380B, Applied Biosystem), according to the manufacturer's protocol.
one-step rt-pcr
Template RNA, 300 µmol/L each of dATP, dGTP, dCTP, and dTTP, 1x
EZ buffer, 2 mmol/L Mn(OAc)2, 0.5 µmol/L each of
primers, and 0.1 µL of rTth polymerase were mixed in a
final volume of 5–50 µL and overlayered with 1 drop of nuclease-free
mineral oil (Sigma). PCR was conducted in a thermal cycler (MJ
Research) with 1 cycle of reverse transcription at 60 °C for 30 min
and 94 °C denaturation for 1 min, followed by 60 cycles of 60 °C
annealing/extension for 1 min and 94 °C of denaturation for 1 min.
After PCR was completed, PCR products were analyzed by 2.0% agarose
gel electrophoresis with 0.5 mg/L ethidium bromide in an
electrophoresis chamber. Photographic images were recorded on Polaroid
667 film.
two-step rt-pcr
Captured mRNA was reverse transcribed to cDNA by the method
described above, using 10 mmol/L dTTP instead of biotin-dUTP. Reactants
were removed by aspiration, and PCR was conducted using either
rTth or Taq polymerase. For PCR with Taq polymerase, buffer
contained 1x GeneAmp PCR buffer (Perkin-Elmer), 1.25 mmol/L
MgCl2, 300 µmol/L each of dATP, dGTP, dCTP, and dTTP, 0.5
µmol/L each of primers, and 0.5 µL of Taq polymerase in a final
volume of 10–50 µL. PCR was conducted in a thermal cycler (MJ
Research) with 30 cycles of 94 °C denaturation for 1 min, 60 °C
annealing for 1 min, and 72 °C extension for 1 min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A, substantial Yoyo-1 fluorescence was obtained from the wells
where >100 ng of mRNA was applied, whereas Yoyo-1 fluorescence was not
increased in the wells of rRNA, tRNA, and DNA even when as much as 10
µg was applied. We have also tested the specificity of cDNA synthesis
on the GenePlate-PP as described in Materials and Methods.
As shown in Fig. 1B
, substantial AttoPhos fluorescence was obtained
from the well where >0.1 ng/well of mRNA was applied but not from the
wells of rRNA, tRNA, and DNA even when as much as 10 µg was applied.
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We then tested the reversibility of mRNA hybridization. After globin mRNA or total liver RNA was applied for hybridization, each well was washed with DEPC water at different temperatures, and the Yoyo-1 assay was conducted. Yoyo-1 fluorescence was reduced to the basal value by addition of boiling DEPC water, whereas ~40–60% of fluorescence remained after a DEPC water wash at room temperature or 55 °C (data not shown).
To assess the hybridization capacity, various amounts of globin mRNA,
total liver RNA, or cell lysates were applied to the GenePlate-PP for
hybridization. Hybridized mRNA was recovered from the plates by adding
boiling water. The buffer concentration was adjusted, and the solution
was applied to a fresh GenePlate-PP for the second hybridization. In
parallel experiments, known concentrations of globin mRNA were also
used as a standard. The cDNA synthesis was conducted in the presence of
biotin-dUTP, and the incorporated biotin was reacted with
streptavidine-alkaline phosphatase followed by AttoPhos detection, as
described in Materials and Methods. The amount of mRNA was
determined by comparison with standard globin mRNA. As shown in Table 1
, ~40–55% of the applied globin mRNA hybridized to the
plates. Applied globin mRNA did not saturate the plates, even when 500
ng was used; 500 ng of globin mRNA is ~1–2 pmol, whereas the amount
of immobilized oligonucleotide was ~20 pmol (data not shown).
Moreover, mRNA capture efficiency was decreased to 8–17% when high
concentrations of total RNA or cell lysate were applied, probably
because high viscosity caused inefficient hybridization.
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We have also analyzed the quality of synthesized cDNA on the plate.
Because first strands of cDNA were covalently attached to the plates
via 5' oligo(dT), we could not dissociate them for quality analysis.
Therefore, second strands of cDNA were synthesized on the plates from
biotin-dUTP or biotin-dATP according to the method described previously
(3)(7). As shown in Fig. 2
, the amount of AttoPhos fluorescence of the second cDNA
synthesis was similar to that of the first cDNA synthesis, suggesting
efficient second cDNA synthesis. Because the second strands of cDNA did
not attach to the plates, the cDNA could be dissociated from the plate
by addition of boiling DEPC water. The resulting cDNA was separated by
agarose gel electrophoresis and transferred to a nylon membrane. As
shown in the inset of Fig. 2
, we could detect a single appropriate band
of globin cDNA, suggesting that synthesized cDNA was full length.
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To conduct quantitative gene expression analysis for molecular
pathological diagnostics, knowledge of well-to-well variation is
crucial (12). Variability of the amounts of immobilized
oligonucleotides (Fig. 3
A, 
), hybridized rabbit globin mRNA (Fig. 3A
, 
), and
synthesized cDNA from captured rabbit globin mRNA (Fig. 3B
, 
) were
all <10–15% within a single microplate (intraassay) or multiple lots
of microplates (interassay). More importantly, the variability of the
amount of PCR products in these intra- and interassay studies was also
within 10% (Fig. 3C
).
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To assess the applicability of GenePlate-PP for molecular pathological
diagnostics, human K562 leukemic cells, which express the b3a2
transcript from the Ph translocation, were treated
with lysis buffer and then centrifuged to remove cell debris and
nuclear DNA. The supernatant solution containing cytosolic mRNA was
applied to the GenePlate-PP for hybridization. After 1 h of
hybridization at room temperature, unbound materials were removed by
washing with hybridization buffer twice. The resulting hybridized mRNA
was used for either measurement of Yoyo-1 or one-step RT-PCR using
rTth polymerase. As a result, we could detect substantial
Yoyo-1 fluorescence (Fig. 4
A) and a PCR band with the expected size of 168 bp (Fig. 4A
, top
inset, indicated by an arrow) from 10 cells. In separate
experiments, hybridized mRNA was converted to cDNA on the microplates,
and the amount of synthesized cDNA was determined by AttoPhos. As shown
in Fig. 4B
, substantial AttoPhos signals were obtained even from as low
as 101
cells, suggesting 100-fold more sensitivity than
Yoyo-1. More interestingly, when PCR was conducted from synthesized
cDNA on the GenePlate-PP, a PCR band was detected from as few as
10 cells (indicated by the arrow in the bottom inset of
Fig. 4A
).
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To determine "false" PCR products from contaminating genomic DNA in
the plates, PCR was conducted with or without reverse transcription. As
shown in Fig. 5
, PCR products of bcr-abl and G3PDH transcripts were
not amplified from the wells of negative reverse transcription. More
interestingly, bcr-abl and G3PDH transcripts were
reamplified from immobilized cDNAs from wells once or twice (Fig. 5
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The plates are not saturated when even as much as 500 ng of
mRNA is applied (which represents ~500 µg of total RNA or
10 cells per well). Because of the power of PCR, this
quantity is more than enough for most experiments.
Another advantage is the strict specificity for mRNA (Fig. 1
), which
eliminates the problem of false PCR amplification from contaminating
genomic DNA, whereas cellulose or beads often contain detectable
amounts of rRNA, tRNA, and DNA. Furthermore, less variation among wells
and plates (Fig. 3
), excellent stability (data not shown), and
availability of various quality control protocols (Fig. 3
)
(12) make this technology very competitive.
Four major techniques are available for immobilizing oligonucleotides onto microplates: physical absorption (13), direct cross-linking to the surface of microplates (14)(15), use of biotin-streptavidin or antigen-antibody reactions (16), and the synthesis of oligonucleotides directly on the surface of microplates (17)(18). These techniques can be applied to PS plates because various functional residues can be induced on the surface of PS plates. However, because of heat instability, PS plates or tubes are not suitable for the 94 °C denaturing step in PCR. Furthermore, because of the high capacity of PS for nonspecific absorption of proteins and DNA/RNA and its sensitivity to organic chemicals (i.e., phenol–chloroform), the majority of molecular biological experiments are carried out in PP microtubes.
Recently, some manufacturers produced PP microplates for molecular biological applications. These microplates allow researchers to conduct high-throughput PCR. However, because of the stable surface characteristics of PP microplates, oligonucleotides cannot be immobilized by conventional methods. In the DNA chip industry, one can synthesize oligonucleotides on PP surfaces (17)(18). However, this requires special instruments. In this study, GenePlate-PP was supplied by AGCT, where manufacturing procedures are licensed from us. The current GenePlate-PP was based on GeNunc PP plates (Nunc); oligonucleotides can be immobilized on any commercially available PP plates, although the amounts of immobilized oligonucleotide and the capacity of mRNA hybridization vary among different manufacturers.
One of the interesting features of PP plates is their fluorescent
characteristics. Although PP plates are cloudy rather than completely
transparent as are PS plates, fluorescence measurement of Yoyo-1 or
equivalent dyes was better performed in PP plates than in PS plates
(10). This allowed us to conduct various analyses easily.
For example, immobilized oligonucleotides (Fig. 3A
), captured mRNA
(Figs. 1A
, 3A
, and 4A
), and synthesized cDNA (Figs. 1B
, 3B
, and 4B
) can
be determined fluorometrically (i.e., without using radioactive
materials).
More interestingly, RT-PCR from synthesized cDNA on the GenePlate-PP
(Two-step RT-PCR, Fig. 4A
, lower inset) was ~100 000-fold more
sensitive than conventional one-step RT-PCR, and the bcr-abl
transcript was detected in cell lysates containing only 100 cells (Fig. 4A
, top inset). This is surprising because two-step RT-PCR required an
inefficient solid phase RT reaction, whereas one-step RT-PCR was
conducted in the more efficient liquid phase reaction by first
dissociating mRNA from the GenePlate-PP. Because we used
rTth for both experiments, the difference was not caused by
the enzyme. Because more primer dimers were formed in one-step RT-PCR
than two-step RT-PCR (Fig. 4A
, both insets), we believe that the
majority of primers are used for dimer formation during RT. In two-step
RT-PCR, these primer dimers are removed, and primers are replenished
when the reaction mixture was switched from cDNA synthesis to PCR.
Furthermore, in the two-step RT-PCR system, microplates can be reused
for multiple PCRs with the same or different primer sets because
synthesized cDNA was covalently attached to the plates at its 5' end.
This is very convenient as a molecular diagnostic tool because we can
repeat PCR reactions in the same well if the results are uncertain.
The Philadelphia chromosome, found frequently in chronic myelogenous leukemia, is a reciprocal translocation of the abl protooncogene from chromosome 9 to a portion of the bcr gene in chromosome 22 [t(9;22)(q34;q11)] (19). Specific RT-PCR amplification of bcr-abl mRNA from peripheral blood cells or bone marrow cells provides a highly sensitive and quantitative methodology for the detection of residual leukemic cells. Because the detection of residual leukemic cells is one of the critical indicators for the treatment of chronic myelogenous leukemia, RT-PCR testing of bcr-abl mRNA is widely available in many institutions. However, in many cases, total RNA or mRNA is first purified from a cell suspension. Using our system, once cell lysates are applied to the GenePlate-PP for hybridization, one can proceed not only with the direct RT-PCR described in this study, but also with Yoyo-1 quantification of total amounts of mRNA (4), which may provide additional means of normalization or quality control of tested materials. A diagnostic assay for bcr-abl is now under test to amplify the transcript from a single leukemic cell consistently from whole blood.
Because of its simplicity and fluorescent characteristics, our GenePlate-PP may be acceptable as a platform for various mRNA expression analyses in basic research, drug screening, and molecular toxicology, as well as molecular pathological diagnostics, with the potential for future automation.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and tRNA (
) were suspended in 50 µL of hybridization buffer
(see Materials and Methods) and applied to the wells of an
oligo(dT)-immobilized PP microplate (GenePlate-PP). After hybridization
at room temperature for 1 h, each well was washed once with
hybridization buffer. (A) Yoyo-1 was diluted in Tris-EDTA to
a final dilution of 1:1000 and applied to each well, and the
fluorescence was determined by a CytoFluor 2300, as described in
Materials and Methods. (B) The contents of each
well were resuspended in 50 µL of cDNA synthesis buffer (see
Materials and Methods) and incubated at 37 °C for 1
h. After each well was washed three times with wash buffer (see
Materials and Methods), 50 µL of wash buffer containing a
1:1000 dilution of streptavidin-alkaline phosphatase conjugate was
added, and the plate was incubated at room temperature for 30 min.
After each well was washed, 50 µL of substrate (AttoPhos) was added,
and the plate was incubated at room temperature for 20 min. The
reaction was terminated by adding an equal volume of 100 mmol/L EDTA,
and the fluorescence was determined by a CytoFluor 2300. Each data
point was the mean ± SD from triplicate determinations.
