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Methods Useful in Genetics and Oncology
ative changes in its expression (ABCAIIPBGD ratio). The advantage of using the
hybridization probe format is its specificity, as confirmed by melting curve analysis for each amplified product, ABCAI and PBGD in our case. Melting curve
analysis at the end of the LightCycler run enables the characterization of the
amplified product and obviates gel electrophoresis. Our data show that standardization with a housekeeping gene (e.g., PBGD) is not completely satisfactory when
the target gene (e.g., ABeAl) is expressed in different tissues and different cells.
Because PBGD is not equally expressed in various tissues, absolute quantification
may be more reliable. However, when we normalized our absolute quantification
of ABCAI mRNA levels with PBGD as a reference gene in the same cell type (e.g.,
fibroblasts, monocytes, HeLa cells) we obtained comparable results. In contrast,
the other commonly used internal standard glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) varies quantitatively in response to various factors making
the interpretation difficult [13,14]. Based on initial experiments with GAPDH as
an endogenous control, we decided to use porphobilinogen deaminase (PBGD) as
the reference gene [15]. Unlike GAPDH, we have amplified both ABCAI and
PBGD transcripts with the same efficiency, which is an important factor for the
final calculation. In contrast to GAPDH, the human PBGD gene is free of pseudogenes [16] and only cDNA-derived products are accumulated during PCR, so that
DNase digestion is not required. We recommend PBGD as a normalization control for mononuclear cells, fibroblasts, adipocytes, HepG2 cells, HeLa cells and
THP-l cells. The two-step quantitative RT-PCR with external standards and
hybridization probes is rapid, reliable, reproducible and not as laborious as other
technologies. The high sensitivity of this method allowed detection and quantification of ABCAI mRNA in a variety of human cells and tissues.
Applications This quantitative method can be used: (a) in the monitoring of drug effects, (b) in
epidemiological studies, (c) in studies assessing correlation between ABeAl
expression and susceptibility to lipid disorders, and (d) in exploring the effects of
polymorphisms in the promoter region of the ABeAl gene.
References
1. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, Drobnik W, Barlage S,
Buchler C, Porch-Ozcurumez M, Kaminski WE, Hahmann HW, Oette K, Rothe G, Aslanidis
C, Lackner K, Schmitz G (1999) The gene encoding ATP-binding cassette transporter 1 is
mutated in Tangier disease. Nat Genet 22:347-351
2. Brooks-Wilson A, Marcil M, Clee S, Zhang LH, Roomp K, vanDam M, Yu L, Brewer C, Collins
JA, Molhuizen HO, Loubser 0, Ouelette BF, Fichter K, Ashobourne-Excoffon KJ, Sensen CW,
Scherer S, Mott S, Denis M, Martindale D, Frohlich J, Morgan K, Koop B, Pimstone S,
Kastelein JJ, Hayden MR (1999) Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 22:336-345
3. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N,
Denefle P, Assman G (1999) Tangier disease is caused by mutations in the gene encoding
ATP-binding cassette transporter 1. Nat Genet 22:352-355
4. Orso E, Broccardo C, Kaminski WE, Bottcher A, Liebisch G, Drobnik W, Gotz A, Chambenoit
0, Diederich W, Langmann T, Spruss T, Luciani MF, Rothe G, Lackner KJ, Chimini G, Schmitz
Methods Useful in Genetics and Oncology
ative changes in its expression (ABCAIIPBGD ratio). The advantage of using the
hybridization probe format is its specificity, as confirmed by melting curve analysis for each amplified product, ABCAI and PBGD in our case. Melting curve
analysis at the end of the LightCycler run enables the characterization of the
amplified product and obviates gel electrophoresis. Our data show that standardization with a housekeeping gene (e.g., PBGD) is not completely satisfactory when
the target gene (e.g., ABeAl) is expressed in different tissues and different cells.
Because PBGD is not equally expressed in various tissues, absolute quantification
may be more reliable. However, when we normalized our absolute quantification
of ABCAI mRNA levels with PBGD as a reference gene in the same cell type (e.g.,
fibroblasts, monocytes, HeLa cells) we obtained comparable results. In contrast,
the other commonly used internal standard glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) varies quantitatively in response to various factors making
the interpretation difficult [13,14]. Based on initial experiments with GAPDH as
an endogenous control, we decided to use porphobilinogen deaminase (PBGD) as
the reference gene [15]. Unlike GAPDH, we have amplified both ABCAI and
PBGD transcripts with the same efficiency, which is an important factor for the
final calculation. In contrast to GAPDH, the human PBGD gene is free of pseudogenes [16] and only cDNA-derived products are accumulated during PCR, so that
DNase digestion is not required. We recommend PBGD as a normalization control for mononuclear cells, fibroblasts, adipocytes, HepG2 cells, HeLa cells and
THP-l cells. The two-step quantitative RT-PCR with external standards and
hybridization probes is rapid, reliable, reproducible and not as laborious as other
technologies. The high sensitivity of this method allowed detection and quantification of ABCAI mRNA in a variety of human cells and tissues.
Applications This quantitative method can be used: (a) in the monitoring of drug effects, (b) in
epidemiological studies, (c) in studies assessing correlation between ABeAl
expression and susceptibility to lipid disorders, and (d) in exploring the effects of
polymorphisms in the promoter region of the ABeAl gene.
References
1. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, Drobnik W, Barlage S,
Buchler C, Porch-Ozcurumez M, Kaminski WE, Hahmann HW, Oette K, Rothe G, Aslanidis
C, Lackner K, Schmitz G (1999) The gene encoding ATP-binding cassette transporter 1 is
mutated in Tangier disease. Nat Genet 22:347-351
2. Brooks-Wilson A, Marcil M, Clee S, Zhang LH, Roomp K, vanDam M, Yu L, Brewer C, Collins
JA, Molhuizen HO, Loubser 0, Ouelette BF, Fichter K, Ashobourne-Excoffon KJ, Sensen CW,
Scherer S, Mott S, Denis M, Martindale D, Frohlich J, Morgan K, Koop B, Pimstone S,
Kastelein JJ, Hayden MR (1999) Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 22:336-345
3. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N,
Denefle P, Assman G (1999) Tangier disease is caused by mutations in the gene encoding
ATP-binding cassette transporter 1. Nat Genet 22:352-355
4. Orso E, Broccardo C, Kaminski WE, Bottcher A, Liebisch G, Drobnik W, Gotz A, Chambenoit
0, Diederich W, Langmann T, Spruss T, Luciani MF, Rothe G, Lackner KJ, Chimini G, Schmitz