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Quantitative analysis of TEL/AML1 fusion transcripts by real-time RT-PCR assay in childhood acute lymphoblastic leukemia

Leukemia volume 14, pages 1526–1528 (2000)Cite this article

Reh cells express the two major fusions transcripts variants resulting from the alternative splicing of AML1 exon 2 at levels similar to those observed in virtually all diagnostic samples so far analyzed. Therefore, we chose total RNA from Reh cell line as a standard for TEL/AML1 quantitation. Quantitation of fusion transcripts in unknown samples can then simply be done by measuring the sample's Ct (threshold cycle) and by using a standard curve constructed either (1) by serial dilutions of 1 μg of Reh cell line RNA-equivalent cDNA into 1× RT buffer or, to reproduce more closely clinical sample analysis, (2) with serial dilutions of 1 μg of Reh cell line RNA in a constant amount of mouse RNA, prior to cDNA synthesis. For each standard curve the amounts of the diluted total Reh RNA or cDNA equivalent varied from 100 to 0.01 ng. In both cases we found a strong linear relationship between the Ct and the starting copy number of the TEL/AML1 transcripts (R2 ⩾ 0.99) over a range of at least four orders of magnitude. Also, the PCR efficiency of TEL/AML1 amplification did not differ significantly with the two methods, as indicated by the slope of the curves obtained with the RNA and cDNA point dilutions (−3.76 and −3.66 corresponding to a PCR efficiency value (E) of 84% and 87% respectively). The TEL/AML1 fusion transcripts could be detected after 104-fold dilution of 1 μg of Reh total RNA in mouse total RNA, which is equivalent to 10 pg of target RNA. The average Ct of the TEL/AML1 target at this dilution was 37.8 with a within-run coefficient of variation (CV) of Ct values varying from 0.8 to 2.4 in different PCR runs. Thus the sensitivity of the method was comparable to that of conventional end-point RT-PCR used for diagnostic purposes. At lower RNA concentration (5 × 104 and 105-fold dilutions), positive samples were still detected but the variation among sample replicates became too high for accurate measurement. Indeed, as the Ct values of TEL/AML1 increased to over 40, the CV among sample replicates was greater than 2.5%, indicating that the quantitation of positive results was not reliable. As the CV for Ct values of triplicates samples indicates the reproducibility of the measure, the limit of accurate quantitation can be easily deduced from the CV.

Variations in starting amount of total RNA or RT efficiency among samples were taken into account by parallel amplification of an endogenous mRNA control. We chose the TBP gene (TATA box-binding protein, a component of the DNA-binding protein complex TFIID) as endogenous control because it is an ubiquitous transcription factor whose expression is constant in normal tissues and is similar to TEL/AML1 expression in the cell line Reh (TBP-Ct and TEL-AML-Ct <22, for 100 ng of total RNA, with a within-run CV 2%). We rejected PBGD gene because of its low expression level compared to TEL/AML1. The endogenous control should be expressed at an adequate level (ideally, at a level equivalent to the standard) in order to avoid underestimating positive samples and obtaining false-negative results. For each experimental sample, the amount of target and endogenous reference was determined from the standard curve. Then the target amount was divided by the endogenous reference amount to obtain a normalized target value.

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Acknowledgements

This work was supported by a grant from the Direction de la Recherche Clinique de l'Assistance Publique-Hôpitaux de Paris (AP-HP), Projet TBI 97-297, France (PB).

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Authors and Affiliations

  1. Laboratoire Central d'Hématologie, Hôpital A Trousseau, Paris, France

    P Ballerini, M Adam & YE Cayre

  2. Service d'Hématologie, Hôpital A Trousseau, Paris, France

    J Landman Parker & G Leverger

  3. Laboratoire de Génétique Moléculaire, Faculté de Sciences Pharmaceutiques et Biologiques de Paris, Paris, France

    I Laurendeau, M Olivi, M Vidaud & I Bièche

  4. Site Transfussionel A Trousseau, Etablissement Français du Sang, Paris, France

    I Gerota

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Ballerini, P., Landman Parker, J., Laurendeau, I. et al. Quantitative analysis of TEL/AML1 fusion transcripts by real-time RT-PCR assay in childhood acute lymphoblastic leukemia. Leukemia 14, 1526–1528 (2000). https://doi.org/10.1038/sj.leu.2401858

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