m Applications in Oncology
esting target for tumor immunotherapy. Clinical trials must however include a
precise monitoring of possible adverse effects on tissue with very low expression
of PRAME. Moreover, PRAME might be a possible marker for minimal residual
disease. Real-time PCR using the LightCycler method allows an accurate quantification of the mRNA expression level of the TAA PRAME in tumors and peripheral blood and bone-marrow samples. Detecting the mRNA expression of this
antigen during the course of chemotherapy and/or hematopoietic transplantation
might constitute a useful tool for the monitoring of minimal residual disease in
AML. Moreover, real-time RT-PCR might be helpful to select potential candidates
for a PRAME specific immunotherapy among AML patients.
Materials
Equipment LightCycler instrument (Roche Diagnostics, Mannheim, Germany)
LightCycler software verso 3.39 (Roche Diagnostics)
LightCycler Capillaries (Roche Diagnostics)
Mac DNASISPro version 3.5 Primer design software (Hitachi)
Reagents
mRNA Quickprep Micro Purification Kit (Amersham Pharmacia Biotech, Little
Chalfont, England, UK)
cDNA Preparation Kit (Superscript II Gibco BRL, Frederick, MA)
Amplification Primers (MWG-Biotech, Munich, Germany)
LightCycler FastStart Kit (Roche Diagnostics)
NuSieve agarose (FMC Bio Products)
Qiaquick PCR Purification Kit (Chatsworth, CA, USA)
Procedure
Cell Culture Cells of the human suspension cell line K562 (a CML cell line established from a
patient with chronic myeloid leukemia in blast crisis) expressed PRAME at a high
level. The cell line was cultured in RPMI 1640 (Biochrom, Berlin, Germany) containing 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and
100 units/ml streptomycin.
Sample Preparation Total RNA was isolated by the phenol/chloroform method (Chomczynski and Sacchi [8]). The mRNA was prepared using the mRNA QuickPrep Micro purification
kit (Amersham Pharmacia Biotech, Little Chalfont, England, UK). In total, 2.5 ~g
mRNA of each sample was transcribed to cDNA (Superscript II Gibco BRL, Frederick, Maryland). For the quantification of the antigen PRAME, the mRNA expression
of PRAME was correlated with the mRNA expression of the house-keeping genes
TBP (IATA-hinding 12rotein) and ~-actin. The primers were used to amplify an
833-bp fragment of the TAA PRAME (Table 1) as described in the literature [4] and
verified with DNASIS software. RT-PCR was performed under the described time
conditions (Table 2), temperatures (Table 3), and with the indicated ingredients.
esting target for tumor immunotherapy. Clinical trials must however include a
precise monitoring of possible adverse effects on tissue with very low expression
of PRAME. Moreover, PRAME might be a possible marker for minimal residual
disease. Real-time PCR using the LightCycler method allows an accurate quantification of the mRNA expression level of the TAA PRAME in tumors and peripheral blood and bone-marrow samples. Detecting the mRNA expression of this
antigen during the course of chemotherapy and/or hematopoietic transplantation
might constitute a useful tool for the monitoring of minimal residual disease in
AML. Moreover, real-time RT-PCR might be helpful to select potential candidates
for a PRAME specific immunotherapy among AML patients.
Materials
Equipment LightCycler instrument (Roche Diagnostics, Mannheim, Germany)
LightCycler software verso 3.39 (Roche Diagnostics)
LightCycler Capillaries (Roche Diagnostics)
Mac DNASISPro version 3.5 Primer design software (Hitachi)
Reagents
mRNA Quickprep Micro Purification Kit (Amersham Pharmacia Biotech, Little
Chalfont, England, UK)
cDNA Preparation Kit (Superscript II Gibco BRL, Frederick, MA)
Amplification Primers (MWG-Biotech, Munich, Germany)
LightCycler FastStart Kit (Roche Diagnostics)
NuSieve agarose (FMC Bio Products)
Qiaquick PCR Purification Kit (Chatsworth, CA, USA)
Procedure
Cell Culture Cells of the human suspension cell line K562 (a CML cell line established from a
patient with chronic myeloid leukemia in blast crisis) expressed PRAME at a high
level. The cell line was cultured in RPMI 1640 (Biochrom, Berlin, Germany) containing 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and
100 units/ml streptomycin.
Sample Preparation Total RNA was isolated by the phenol/chloroform method (Chomczynski and Sacchi [8]). The mRNA was prepared using the mRNA QuickPrep Micro purification
kit (Amersham Pharmacia Biotech, Little Chalfont, England, UK). In total, 2.5 ~g
mRNA of each sample was transcribed to cDNA (Superscript II Gibco BRL, Frederick, Maryland). For the quantification of the antigen PRAME, the mRNA expression
of PRAME was correlated with the mRNA expression of the house-keeping genes
TBP (IATA-hinding 12rotein) and ~-actin. The primers were used to amplify an
833-bp fragment of the TAA PRAME (Table 1) as described in the literature [4] and
verified with DNASIS software. RT-PCR was performed under the described time
conditions (Table 2), temperatures (Table 3), and with the indicated ingredients.