John M. S. Bartlett.pdf - Bio-Nica.info

PCR Detection of Tumor Cells 187
8. Denaturating agarose gels (1.2%) containing 2.2 mM formaldehyde for RNA.
9. PCR reagents and primers for amplifying reference genes like β-actin or GAPDH.
2.2. Consensus RT-PCR
1. GeneAmp RNA PCR Core Kit (Perkin–Elmer, Weiterstadt, Germany).
2. Amplitaq Gold DNA polymerase with GeneAmp 10× PCR buffer II (Perkin–Elmer) and
MgCl 2 solution.
3. Mixture of dATP, dCTP, dGTP, and dTTP, concentration 2.5 mM each (stock solution
100 mM, Promega, Madison WI).
4. Consensus primers FR1C (5′-GGTGCAGCTGS(A/T)GSAGTC(A/G/T)GG-3′) (5), FR3A
(5′-ACACGGCYSTGTATTACTGT-3′) (6), and LJH (5′-TGAGGAGACGGTGACC-3′) (6).
5. Ethidium bromide-stained agarose gels (2 and 5%, NuSieve 3:1 Agarose, FMC/Biozym,
Oldendorf, Germany).
2.3. Identification of the Myeloma CDR Regions
1. Scalpel.
2. DNA purification kit (EasyPure DNA Purification kit, Biozym, Göttingen, Germany).
3. Cloning kit for PCR products (TOPO TA Cloning Kit, Invitrogen, De Schelp, The
Netherlands).
4. LB medium, LB plates, with 50 µg/mL kanamycin (Boehringer Mannheim, Mannheim,
Germany).
5. Lysis buffer (100 µg/mL of Proteinase K (Boehringer Mannheim) in 10 mM Tris-Cl,
pH 7.5, and 1 mM EDTA).
6. PCR reagents (see Subheading 2.2.) and M13 universal (forward) and M13 reverse
primers.
7. Sequitherm Cycle Sequencing kit (Biozym, Göttingen, Germany).
2.4. Quantitative PCR (qPCR) Using ASO Primers
1. DNA processing software, for example Gene Jockey II (Biosoft, Cambridge, United
Kingdom).
2. PCR reagents, ASO primers as designed, LJH primer, ethidium bromide stained agarose
gels (2% or 5%).
3. Buffy coat DNA from healthy donors.
4. Computer program for likelihood maximization and χ 2 minimization, for example,
MAXLIKE. This program written in C (Watcom C/C++ version 10.6, Powersoft, Waterloo,
Canada) running under DOS on an IBM compatible PC or the source code can be
obtained for free according to the terms and conditions for copying, distribution, and
modification of programs under the GNU general public license agreement from the
authors (see contact address).
5. Myeloma or B-cell lines, for example U266 (10) or Daudi (11).
3. Methods
3.1. Isolation of Nucleic Acids
1. Collect bone marrow aspirate in two 10-mL syringes with heparin added for anticoagulation.
This will be the starting material for both the identification of the VDJ sequence of
the malignant clone and the testing of ASO primers.
2. Isolate mononucleated cells by centrifugation over Ficoll separating solution.
3. Wash cells twice in phosphate-buffered saline, pelleting between washes by centrifugation
at 800g for 10 min.