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The use of housekeeping genes for real-time PCR-based quantification of fusion gene transcripts in acute myeloid leukemia

Leukemia volume 18, pages 1551–1553 (2004)Cite this article

TO THE EDITOR

Quantification of minimal residual disease (MRD) during and after antileukemic therapy is of growing interest in the treatment of acute myeloid leukemia (AML). Real-time reverse transcriptase polymerase chain reaction (PCR) is a widely used method for detection and quantification of MRD. Commonly used targets for MRD diagnostics in AML are fusion transcripts, including AML1-ETO, CBFB-MYH11 and PML-RARA. Recent studies have shown that it is possible to detect molecular relapse of AML before hematologic manifestation on the basis of increased ratios of fusion transcripts. In addition, it was possible to define threshold values at certain time points that predict relapses.1 Quantitative RNA expression studies are usually carried out relative to the expression of a housekeeping gene as endogenous control to compensate for uneven cell numbers, RNA quality, or variations in reverse transcription efficiancies, of individual samples. The appropriate control gene for quantitative assessment should meet certain criteria. These include constitutive expression in all cell types at relative stable levels, stability and expression level comparable to the target gene. In addition, housekeeping genes with known existing pseudogenes should be neglected to avoid the amplification of genomic DNA. Housekeeping genes comprise a wide variety of genes that mainly code for proteins that are essential for the cell function.2 Some of these genes code for components of the cytoskeleton (β-actin, α-tubulin), the major histocompatibility complex (B2M), enzymes of metabolic pathways (glycerolaldehydphosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PDH), porphobilinogen deaminase (PBGD)) or enzymes of signaling pathways (ABL). Recently, some reports demonstrated that the expression of housekeeping genes varied considerably under experimental circumstances.3 Therefore, the choice of an appropriate housekeeping gene as endogenous control is essential for the accuracy and reliability of a quantitative PCR approach. We quantitatvely analyzed the expression of AML1-ETO, CBFB-MYH11 and PML-RARA in AML samples relative to four different housekeeping genes – B2M, PBGD, G6PDH and ABL – by real-time reverse transcription (RT)-PCR using the LightCycler System. For all analyses, mRNA was automatically isolated by the MagnaPureLC (Roche, Mannheim, Germany), and cDNA was synthesized by random primed RT using Superscript II (Invitrogen Karlsruhe, Germany). Quantification of target and control genes was performed using standard curve analysis. The PCR protocols for AML1-ETO, CBFB-MYH11 and PML-RARA as well as G6PDH and ABL have been published,1, 4 while B2M and PBGD were quantified using the housekeeping gene kit B2M and PBGD, according to the manufacturer's protocol (Roche Diagnostics, Mannheim, Germany). Bone marrow samples of AML patients were analyzed at diagnosis (n=29) and in follow-up samples (n=21) during and after cytostatic therapy. In addition, the expression of housekeeping genes was analyzed in the peripheral blood of healthy volunteers (n=14).

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References

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Authors and Affiliations

  1. Department of Internal Medicine III, University of Munich, Klinikum Grosshadern, Munich, Germany

    M Weisser, T Haferlach, C Schoch, W Hiddemann & S Schnittger

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  1. M Weisser
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  2. T Haferlach
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  3. C Schoch
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  4. W Hiddemann
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  5. S Schnittger
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Correspondence to M Weisser.

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Weisser, M., Haferlach, T., Schoch, C. et al. The use of housekeeping genes for real-time PCR-based quantification of fusion gene transcripts in acute myeloid leukemia. Leukemia 18, 1551–1553 (2004). https://doi.org/10.1038/sj.leu.2403438

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