mil Applications in Oncology
Materials
Equipment LightCycler instrument
Reagents Isogen RNA extraction buffer (Nippon Gene, Tokyo, Japan)
Glycogen (Roche Diagnostics, Mannheim, Germany)
Random hexanucleotide primer (Pharmacia Biotech, Uppsala, Sweden)
SuperScript II RNase H-reverse transcriptase (Gibco BRL, Gaithersburg, USA)
Amplification primer (Nihon Gene Research Laboratories, Sendai, Japan)
Hybridization probes (Nihon Gene Research Laboratories)
LightCycler-DNA Master hybridization probes (Roche Diagnostics)
TaqStart antibody (Clontech, Palo Alto, USA)
Procedure
Sample preparation Peritoneal washes (50-100 ml) obtained from the Douglas cavity at the beginning
of each operation were centrifuged at 1,800 rpm for 5 min to collect intact cells,
rinsed with PBS and dissolved in 1 ml Isogen. Total RNA was extracted using a
guanidinium-isothiocyanate-phenol-chloroform-based method in the presence of
glycogen (20 Ilg/sample). Extracted total RNA up to 5 Ilg were preincubated with
50 ng of random hexanucleotide primer in 9 fll of solution for 10 min at 70°C. After
chilling on ice, the following were added: 4 fll of fivefold synthesis buffer (250 mM
Tris-HCI, pH 8.3,375 mM KCI, 15 mM MgCI2), 2 fll of 100 mM dithiothreitol,4 fll of
2.5 mM of each dNTP and 1 fll of SuperScript II RNase Wreverse transcriptase
(200 units/fll). The reaction mixture was preincubated for 10 min at 25°C and then
incubated for 50 min at 42°C, followed by heating at 70°C for 15 min. The resultant
first-strand cDNA was used for PCR amplification with the LightCycler instrument.
Oligonudeotides The CEA-specific oligonucleotide primers and hybridization probes used are
designed with the aid of Oligo primer analysis software based on published
sequences [5] and synthesized by Nihon Gene Research Laboratories. Details for
primers and probes used in this study are summarized in Table 1. To quantify and
Table 1. Oligonucleotides
Carcinoembryonic antigen (GenBank Accession #M29540)
Sequence
5' -Position
Le ngth
GC(%) Tm (0C)
Primers
AACTTCTCCTGGTCTCTCAGCT
2148
22
50
63.9
GCAAATGCTTTAAGGAAGAAG
2272
21
38
6 1.9
Product
2148-2272
125 bp
Hybridization probes
TGAAf>JGAAGAAACTACACCAGG-P
2228
23
39
63.9
LCRed640-CTGCTATATCAGAGCAACCCCAA-P 2204
23
48
68.6
Materials
Equipment LightCycler instrument
Reagents Isogen RNA extraction buffer (Nippon Gene, Tokyo, Japan)
Glycogen (Roche Diagnostics, Mannheim, Germany)
Random hexanucleotide primer (Pharmacia Biotech, Uppsala, Sweden)
SuperScript II RNase H-reverse transcriptase (Gibco BRL, Gaithersburg, USA)
Amplification primer (Nihon Gene Research Laboratories, Sendai, Japan)
Hybridization probes (Nihon Gene Research Laboratories)
LightCycler-DNA Master hybridization probes (Roche Diagnostics)
TaqStart antibody (Clontech, Palo Alto, USA)
Procedure
Sample preparation Peritoneal washes (50-100 ml) obtained from the Douglas cavity at the beginning
of each operation were centrifuged at 1,800 rpm for 5 min to collect intact cells,
rinsed with PBS and dissolved in 1 ml Isogen. Total RNA was extracted using a
guanidinium-isothiocyanate-phenol-chloroform-based method in the presence of
glycogen (20 Ilg/sample). Extracted total RNA up to 5 Ilg were preincubated with
50 ng of random hexanucleotide primer in 9 fll of solution for 10 min at 70°C. After
chilling on ice, the following were added: 4 fll of fivefold synthesis buffer (250 mM
Tris-HCI, pH 8.3,375 mM KCI, 15 mM MgCI2), 2 fll of 100 mM dithiothreitol,4 fll of
2.5 mM of each dNTP and 1 fll of SuperScript II RNase Wreverse transcriptase
(200 units/fll). The reaction mixture was preincubated for 10 min at 25°C and then
incubated for 50 min at 42°C, followed by heating at 70°C for 15 min. The resultant
first-strand cDNA was used for PCR amplification with the LightCycler instrument.
Oligonudeotides The CEA-specific oligonucleotide primers and hybridization probes used are
designed with the aid of Oligo primer analysis software based on published
sequences [5] and synthesized by Nihon Gene Research Laboratories. Details for
primers and probes used in this study are summarized in Table 1. To quantify and
Table 1. Oligonucleotides
Carcinoembryonic antigen (GenBank Accession #M29540)
Sequence
5' -Position
Le ngth
GC(%) Tm (0C)
Primers
AACTTCTCCTGGTCTCTCAGCT
2148
22
50
63.9
GCAAATGCTTTAAGGAAGAAG
2272
21
38
6 1.9
Product
2148-2272
125 bp
Hybridization probes
TGAAf>JGAAGAAACTACACCAGG-P
2228
23
39
63.9
LCRed640-CTGCTATATCAGAGCAACCCCAA-P 2204
23
48
68.6