Detection of Exosomal PML-RARA Fusion Gene Expression Level by Droplet Digital PCR / 中国实验血液学杂志
Journal of Experimental Hematology
; (6): 747-752, 2019.
Article
in Zh
| WPRIM
| ID: wpr-771890
Responsible library:
WPRO
ABSTRACT
OBJECTIVE@#To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR).@*METHODS@#By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique.@*RESULTS@#The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected.@*CONCLUSION@#A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.
Full text:
1
Database:
WPRIM
Main subject:
Leukemia, Promyelocytic, Acute
/
Gene Expression
/
Oncogene Proteins, Fusion
/
Polymerase Chain Reaction
/
Protein Isoforms
/
Exosomes
Type of study:
Diagnostic_studies
Limits:
Humans
Language:
Zh
Journal:
Journal of Experimental Hematology
Year:
2019
Document type:
Article