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Determination of PML-RARalpha gene expression by using the RT-qPCR technique from blood cells RNA in murine model of acute promyelocytic leucemia
Švec, David ; Semecký, Vladimír (advisor) ; Herink, Josef (referee)
Our goal was to develop an accurate, quick and affordable method for following up the progression of acute promyelocytic leucemia in murine model. We took advantage of a developed system of reverse transkriptase quantitative polymerase chain reaction (RT-qPCR) performinng human bone marrow samples to develop a quantitative method of measurement of PML-RARalpha transcripts for murine model. PML-RARalpha transcripts are hallmarks of acute promyelocytic leucemia (APL) with t(15;17) translocation. For measuring bcr1 transcripts, RNA was isolated from murine blood. We present here a method for quantification of t(15;17) transcript bcr1, which is developed in myeloid cells of transgenic mice and then is injected into FVB/N mice. The expression of PML-RARalpha transcripts is normalized using the housekeeping 18S ribosomal subunit gene. We have quantitative data for three healthy mice (FVB/N) and for six ill mice (APL), which were injected 104, 103 or 102 blasts. We show negative expression in FVB/N mice and different copy numbers for APL mice. There is not shown a complete correlation between the amount of injected cells and the amount of transkript copies.
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Single cell gene expression profiling and quality control
Švec, David ; Kubista, Mikael (advisor) ; Beneš, Vladimír (referee) ; Vopálenský, Václav (referee)
Single cell gene expression profiling and quality control David Švec Institute of Biotechnology AS CR, Laboratory of Gene Expression, Prague, Czech Republic TATAA Biocenter, Research & Development, Gothenburg, Sweden Abstract: Gene expression profiling has become an exceedingly important tool for describing occurence of mRNA in tissue samples and even single cells. Most often we use it for characterization of cell types, degree of differentiation and pathology on a molecular level. In our newly established laboratory, we developed high resolution qPCR tomography to show distribution of tens of maternal mRNAs within a single oocyte. We demonstrated that distribution of mRNAs has an important role in further development of the organism. For high resolution qPCR tomography, where one oocyte is divided in tens of samples and about fifty genes are studied in each sample, we optimized dye based protocol for microfluidic high-throughput platform BioMark. Next step was complementing the molecular profile of tens most important genes with information about histology of each selected tissue section using laser microdissection. As a model we used embryonic development of mouse molar. Our goal was to describe interaction of up to one hundred genes in different stages of development and on the single cell level. This...
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Determination of PML-RARalpha gene expression by using the RT-qPCR technique from blood cells RNA in murine model of acute promyelocytic leucemia
Švec, David ; Semecký, Vladimír (advisor) ; Herink, Josef (referee)
Our goal was to develop an accurate, quick and affordable method for following up the progression of acute promyelocytic leucemia in murine model. We took advantage of a developed system of reverse transkriptase quantitative polymerase chain reaction (RT-qPCR) performinng human bone marrow samples to develop a quantitative method of measurement of PML-RARalpha transcripts for murine model. PML-RARalpha transcripts are hallmarks of acute promyelocytic leucemia (APL) with t(15;17) translocation. For measuring bcr1 transcripts, RNA was isolated from murine blood. We present here a method for quantification of t(15;17) transcript bcr1, which is developed in myeloid cells of transgenic mice and then is injected into FVB/N mice. The expression of PML-RARalpha transcripts is normalized using the housekeeping 18S ribosomal subunit gene. We have quantitative data for three healthy mice (FVB/N) and for six ill mice (APL), which were injected 104, 103 or 102 blasts. We show negative expression in FVB/N mice and different copy numbers for APL mice. There is not shown a complete correlation between the amount of injected cells and the amount of transkript copies.
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