微滴式数字PCR技术检测外泌体PML-RARA融合基因的表达
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摘要
目的:利用微滴式数字PCR技术检测外泌体中PML-RARA融合基因表达水平。方法:使用基于Taqman探针法的ddPCR技术,建立可检测长型和短型PML-RARA融合基因转录本的方法。以PML-RARA阴性细胞株HL-60细胞的RNA逆转录合成的cDNA为阴性对照建立空白检测限(LOB);以表达长型PML-RARA融合基因的NB4细胞RNA逆转录合成的cDNA和含短型PML-RARA cDNA的重组质粒为模板分别确定两者的最低检测限(LOD);并检测NB4细胞培养上清来源的外泌体和急性早幼粒白血病患者血清来源的外泌体中PML-RARA融合基因的表达情况。结果:采用ddPCR技术针对长型和短型PML-RARA转录本测得的LOB分别为每微升PCR反应体系中含0.0725拷贝和0.083拷贝;测得的LOD分别为每微升PCR反应体系中含0.19拷贝和0.21拷贝;使用该检测方法,在NB4细胞培养上清来源的外泌体和急性早幼粒细胞白血病患者血清来源的外泌体中均可检测到PML-RARA融合基因的表达。结论:建立了一种基于ddPCR技术检测融合基因转录本的方法,可对外泌体中的微量PML-RARA转录本进行绝对定量,为无创动态监测急性早幼粒细胞白血病患者血清外泌体中PML-RARA融合基因的表达水平提供了可能。
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.
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.
引文
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11王军,郭卫.外泌体检测在骨肿瘤诊疗中的意义.中国肿瘤临床,2018;45(12):648-651.
12 Goldvaser H,Gutkin A,Beery E,et al.Characterisation of bloodderived exosomal hTERT mRNA secretion in cancer patients:Apotential pan-cancer marker.Br J Cancer,2017;117(3):353-357.
13 Bernard V,Kim DU,San Lucas FA,et al.Circulating nucleic acids associate with outcomes of patients with pancreatic cancer.Gastroenterology,2019;156(1):108-118.
14 Cicconi L,Fenaux P,Kantarjian H,et al.Molecular remission as a therapeutic objective in acute promyelocytic leukemia.Leukemia,2018;32(8):1671-1678.
15 van der Velden VH,Panzer-Grumayer ER,Cazzaniga G,et al.Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting.Leukemia,2007;21(4):706-713.
16 Whale AS,Huggett JF,Cowen S,et al.Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.Nucleic Acids Res,2012;40(11):e82.
17 Hindson BJ,Ness KD,Masquelier DA,et al.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.Anal Chem,2011;83(22):8604-8610.
2 Albano F,Zagaria A,Anelli L,et al.Absolute quantification of the pretreatment PML-RARA transcript defines the relapse risk in acute promyelocytic leukemia.Oncotarget,2015;6(15):13269-13277.
3 Brunetti C,Anelli L,Zagaria A,et al.Droplet digital PCR is a reliable tool for monitoring minimal residual disease in acute promyelocytic leukemia.J Mol Diagn,2017;19(3):437-444.
4 Atretkhany KN,Drutskaya MS,Nedospasov SA,et al.Chemokines,cytokines and exosomes help tumors to shape inflammatory microenvironment.Pharmacol Ther,2016;168:98-112.
5 Wu L,Zhang X,Zhang B,et al.Exosomes derived from gastric cancer cells activate NF-kappaB pathway in macrophages to promote cancer progression.Tumour Biol,2016;37(9):12169-12180.
6 Mao L,Li X,Gong S,et al.Serum exosomes contain ECRG4 mRNAthat suppresses tumor growth via inhibition of genes involved in inflammation,cell proliferation,and angiogenesis.Cancer Gene Ther,2018;25(9-10):248-259.
7 Simons M,Raposo G.Exosomes--vesicular carriers for intercellular communication.Curr Opin Cell Biol,2009;21(4):575-581.
8 Hindson CM,Chevillet JR,Briggs HA,et al.Absolute quantification by droplet digital PCR versus analog real-time PCR.Nat Methods,2013;10(10):1003-1005.
9 Gabert J,Beillard E,van der Velden VH,et al.Standardization and quality control studies of'real-time'quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia-a Europe Against Cancer program.Leukemia,2003;17(12):2318-2357.
10 Trams EG,Lauter CJ,Salem NJ,et al.Exfoliation of membrane ecto-enzymes in the form of micro-vesicles.Biochim Biophys Acta,1981;645(1):63-70.
11王军,郭卫.外泌体检测在骨肿瘤诊疗中的意义.中国肿瘤临床,2018;45(12):648-651.
12 Goldvaser H,Gutkin A,Beery E,et al.Characterisation of bloodderived exosomal hTERT mRNA secretion in cancer patients:Apotential pan-cancer marker.Br J Cancer,2017;117(3):353-357.
13 Bernard V,Kim DU,San Lucas FA,et al.Circulating nucleic acids associate with outcomes of patients with pancreatic cancer.Gastroenterology,2019;156(1):108-118.
14 Cicconi L,Fenaux P,Kantarjian H,et al.Molecular remission as a therapeutic objective in acute promyelocytic leukemia.Leukemia,2018;32(8):1671-1678.
15 van der Velden VH,Panzer-Grumayer ER,Cazzaniga G,et al.Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting.Leukemia,2007;21(4):706-713.
16 Whale AS,Huggett JF,Cowen S,et al.Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.Nucleic Acids Res,2012;40(11):e82.
17 Hindson BJ,Ness KD,Masquelier DA,et al.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.Anal Chem,2011;83(22):8604-8610.