Development of a biochip‐based assay integrated in a global strategy for identification of fusion transcripts in acute myeloid leukemia: a work flow for acute myeloid leukemia diagnosis

Giusiano, Sophie; Formisano-Tréziny, Christine; Benziane, A.; Maroc, N; Picard, Christophe; Hermitte, Fabienne; Taranger-Charpin, Colette; Gabert, Jean

doi:10.1111/j.1751-553x.2009.01201.x

Cited by 4 publications

(5 citation statements)

References 38 publications

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“…Compared with the previous reports [11–14, 25], the procedure established here had many advantages. First, the procedure here targeted more chromosomal translocations.…”

Section: Discussionmentioning

confidence: 95%

“…However, the previous reports only had allowed the analysis of only a few rearrangements and one split variant in each fusion gene. For instance, the AML Fusion Chip [14] only included three major types of rearrangements in AML: AML1-ETO , CBFB-MYH11 , and 11q23/ MLL abnormalities ( MLL-AF9 , MLL-ENL , MLL-AF6 , and MLL-AF10 ); the gel-based biochip developed by Nasedkina et al [12] addressed 13 fusion variants in 7 leukemia translocations. Second, the range of detection was wider, covering AML, ALL, and CML.…”

Section: Discussionmentioning

confidence: 99%

“…Microarray is another useful detection assay. Two biochip-based diagnostic systems were reported: a gel-based biochip by Nasedkina et al [11, 12] and MLL Fusion Chip and AML Fusion Chip from France [13, 14]. In these previous works, the gel-based biochip only targeted 7 chromosomal translocations, addressing 13 fusion variants in sum, while other two chips covered certain leukemia group.…”

Section: Introductionmentioning

confidence: 99%

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A Pipeline with Multiplex Reverse Transcription Polymerase Chain Reaction and Microarray for Screening of Chromosomal Translocations in Leukemia

Xiong

Zhang

et al. 2013

BioMed Research International

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Chromosome rearrangements and fusion genes present major portion of leukemogenesis and contribute to leukemic subtypes. It is practical and helpful to detect the fusion genes in clinic diagnosis of leukemia. Present application of reverse transcription polymerase chain reaction (RT-PCR) method to detect the fusion gene transcripts is effective, but time- and labor-consuming. To set up a simple and rapid system, we established a method that combined multiplex RT-PCR and microarray. We selected 15 clinically most frequently observed chromosomal rearrangements generating more than 50 fusion gene variants. Chimeric reverse primers and chimeric PCR primers containing both gene-specific and universal sequences were applied in the procedure of multiplex RT-PCR, and then the PCR products hybridized with a designed microarray. With this approach, among 200 clinic samples, 63 samples were detected to have gene rearrangements. All the detected fusion genes positive and negative were validated with RT-PCR and Sanger sequencing. Our data suggested that the RT-PCR-microarray pipeline could screen 15 partner gene pairs simultaneously at the same accuracy of the fusion gene detection with regular RT-PCR. The pipeline showed effectiveness in multiple fusion genes screening in clinic samples.

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“…Compared with the previous reports [11–14, 25], the procedure established here had many advantages. First, the procedure here targeted more chromosomal translocations.…”

Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Pipeline with Multiplex Reverse Transcription Polymerase Chain Reaction and Microarray for Screening of Chromosomal Translocations in Leukemia

Xiong

Zhang

et al. 2013

BioMed Research International

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“…Introduction of real-time qPCR has significantly improved the use of molecular biomarkers for diagnosis/prognosis of human diseases and monitoring therapeutic effects and patient follow-up, 23 even in cases of rare pathologies. 25 qPCR has become the method of choice because of its reliability, high sensitivity, good reproducibility, and wide dynamic quantification range 26 with a small amount of starting material. Another specific advantage of qPCR for diagnostic applications is that no post-PCR steps are re-quired, thus avoiding the possibility of cross-contamination due to PCR products.…”

Section: Discussionmentioning

confidence: 99%

Development of Plasmid Calibrators for Absolute Quantification of miRNAs by Using Real-Time qPCR

Formisano-Tréziny

Feliciano

Gabert

2012

The Journal of Molecular Diagnostics

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MicroRNAs (miRNAs) are small noncoding RNAs of approximately 18 to 25 nucleotides in length that negatively regulate gene expression via either the degradation or translational inhibition of their target mRNAs. Because miRNAs are essential for the regulation of critical physiological processes as well as a variety of pathological events, they have emerged as a novel class of molecular diagnostic biomarkers and therapeutic agents or targets. Accordingly, the need for novel methods for the quantification of miRNA has increased due to interest in their clinical implications. Currently, real-time quantitative polymerase chain reaction (qPCR) is considered the most robust technology for nucleic acid quantification. Different tools for miRNA quantification by using qPCR are now commercially available, but only relative quantification strategies have been reported. This situation may be partly due to the difficulty in obtaining an appropriate molecule with which to establish an miRNA calibration range. Here, we describe a rapid and convenient strategy for the development of a calibrator, which enables the absolute quantification of miRNAs by using qPCR and allows the cloning of a synthetic sequence of interest instead of a PCR product into a plasmid.

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“…Some studies have been based on electronic hybridization and fluorescence detection or biochips. 13,14 Several multiplex RT-PCR systems have been described for detection of some of these fusion transcripts, but often using analysis of the amplified fragments by agarose gel electrophoresis stained with ethidium bromide. 15,16 For example, Pallisgaard et al 17 proposed a protocol based on eight multiplex RT-PCRs for screening of 29 acute leukemia chromosomal translocations known at the time of the publication; however, these orientation multiplex RT-PCRs had to be followed by different nested PCRs, specific for the suspected fusion transcripts, and had to be confirmed by sequencing.…”

mentioning

confidence: 99%

Design and Feasibility of a Novel, Rapid, and Simple Fluorescence 26-Plex RT-PCR Assay for Simultaneous Detection of 24 Fusion Transcripts in Adult Acute Myeloid Leukemia

Laforêt¹,

Turlure²,

Lippert³

et al. 2013

The Journal of Molecular Diagnostics

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Identification of chromosomal abnormalities is mandatory for classification of acute myeloid leukemia (AML), and the abnormalities have to be determined quickly, to allow patient enrollment in multicenter protocols and/or for selecting therapeutic strategies. Rapid AML molecular diagnosis is often difficult to achieve, however, because it is based on numerous different RT-PCR protocols. We developed a new RT-PCR method, one that does not require a nested step, to simultaneously detect all AML fusion transcripts from six major recurrent translocations found in adults: t(15;17)(q22;q12), inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], t(8;21)(q22;q22), t(6;9)(p23;q34), t(9;22)(q34;q11), and t(10;11)(p13;q14). Specific primers for RT-PCR detection of the 24 fusion transcripts, along with two transcripts for controls, were designed for this 26-plex RT-PCR. Each PCR product had a different size and was separated by capillary electrophoresis. We also designed a multiplex positive control with 24 chimeric RNAs, corresponding to all chimeric RNAs tested. Compared with classical molecular biology protocols and cytogenetic analyses used as reference standards, results of the 26-plex RT-PCR method were concordant in all 204 (100%) cases of adult AML tested. Results were obtained in less than 24 hours. Because of the multiplex positive control, interpretation of the peaks was very easy, without any ambiguity. The tumor cell detection threshold was 1.5%.

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Development of a biochip‐based assay integrated in a global strategy for identification of fusion transcripts in acute myeloid leukemia: a work flow for acute myeloid leukemia diagnosis

Cited by 4 publications

References 38 publications

A Pipeline with Multiplex Reverse Transcription Polymerase Chain Reaction and Microarray for Screening of Chromosomal Translocations in Leukemia

A Pipeline with Multiplex Reverse Transcription Polymerase Chain Reaction and Microarray for Screening of Chromosomal Translocations in Leukemia

Development of Plasmid Calibrators for Absolute Quantification of miRNAs by Using Real-Time qPCR

Design and Feasibility of a Novel, Rapid, and Simple Fluorescence 26-Plex RT-PCR Assay for Simultaneous Detection of 24 Fusion Transcripts in Adult Acute Myeloid Leukemia

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