Detection of circulating hepatocellular carcinoma cells in peripheral venous blood by reverse transcription-polymerase chain reaction

Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral blood (5 ml) samples were ob-tained from 93 patients with hepatocellular carcinoma(HCC) and from 33 control subjects (9 with liver cir-rhosis after hepatitis B,14 with chronic hepatitis B,10with normal liver function). To identify HCC cells inperipheral blood, liver-specific human alpha-fetopro-tein (AFP) mRNA was amplified from total RNA ex-tracted from whole blood by reverse transcription-polymerase chain reaction.Results: AFPmRNA was detected in 50 blood samplesfrom the HCC patients (50/93, 53.8%). In contrast,there were no clinical control patients whose samplesshowed detectable AFPmRNA in PBL. The presence ofAFPmRNA in blood seemed to be correlated with thestage (by TNM classification) of HCC, the serum AFPvalue, and the presence of intrahepatic metastasis,portal vein thrombosis, tumor diameter and/or distantmetastasis. In addition, AFPmRNA was detected in theblood of 21 patients with metastasis at extrahepaticorgans (100%) in contrast to 29 (40.3%)of 72 pa-tients without metastasis.Conclusion: The presence of AFPmRNA in peripheralblood may be an indicator of malignant hepatocytes,which might predict hematogenous spreading metasta-sis of tumor cells in patients with HCC.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

Positive reverse transcription-polymerase chain reaction assay results in patients recovered from COVID-19: Report of two cases

BACKGROUND Coronavirus disease 2019(COVID-19)has spread around the globe.On February 28,2020,the World Health Organization adjusted the risk of spread and impact of COVID-19 to“very high”at the global level.Studies have mainly focused on the etiology,epidemiology,and treatment of COVID-19 to limit further spread and the negative impact of the disease,while less attention has been devoted to the follow-up and reexamination of patients who recovered from COVID-19 or were released from quarantine.CASE SUMMARY This study reports two cases where patients who had negative reverse transcription-polymerase chain reaction(RT-PCR)test results and met the criteria for discharge subsequently had positive RT-PCR test results.The clinical manifestations and computed tomography(CT)findings of these patients were examined.The conversion of RT-PCR test results in these two patients may be related to false-negative and false-positive outcomes of the test.CT images helped track improvement of pulmonary lesions.CONCLUSION The timing of discharge of COVID-19 patients should be determined by comprehensive analysis of CT images and RT-PCR test results.

Quantification of Porcine Follicle-stimulating Hormone Receptor Messenger Ribonucleic Acid by Reverse Transcription competitive Polymerase Chain Reaction

An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.

Detection of hepatocellular carcinoma cells in the peripheral blood with reverse--transcription polymerase chain reaction

In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.

Quantification of mRNA Levels by Fluorescently Labelled Reverse Transcription Competitive PCR

A reproducible,quantitative,non-radioactive method for the analysis of mRNA expression is described.After RNA preparation and cDNA synthesis,the cDNA was co-amplified with an internal standard in the same PCR system.The PCR products containing both targen and internal standard amplificates were electrophoresed and detected on an ABI 377 DNA Sequencer.For each sample,β-actin was also quantified by an identical procedure to compensate for relative differences between samples in the integrity of the individual RNA samples and for variations in reverse transcription.Due to the linear relationship between cDNA content and PCR product ratio of target cDNA template and competitive standard,a single PCR reaction was sufficient for quantification of a sample.The experimental results showed that the method is a mRNA quantitative RT-PCR method with high sensitivity and good reproducibility.It can be used in large-scale accurate quantitative analyses of mRNA expression of any gene.

RT-PCR技术检测尿液中survivin mRNA对膀胱癌诊断价值的系统评价

背景与目的:Survivin基因逐渐成为肿瘤诊断、判断预后和治疗的靶点。用RT-PCR技术检测尿液中survivin mRNA作为一种无创性手段用于膀胱癌的诊断,国内外相关研究较多,但其结果不一。本研究旨在应用系统评价方法对相关文献进行客观评价,以评估RT-PCR技术检测尿液中survivin mRNA对膀胱癌的诊断价值。方法:以膀胱肿瘤、生存素、逆转录-聚合酶链反应、敏感度、特异度、诊断等为主要检索词检索PubMed、EMBASE、SCI、Cochrane Library、中国生物医学文献数据库(CBM)、中国期刊全文数据库(CJFD)、中文科技期刊数据库(CSJD)、中华医学会数字化期刊等数据库,并结合Google Scholar等搜索引擎全面搜集1997年到2009年4月关于RT-PCR检测尿液survivin mRNA诊断膀胱癌的研究。根据QUADAS(quality assessment of diagnostic accuracy studies)质量评价标准评价纳入文献质量,用Meta-Disc软件对结果指标进行分析。结果:最终纳入26个研究,共2416例。Meta分析结果显示,RT-PCR技术检测尿液中survivin mRNA诊断膀胱癌与病理检查相比准确度指标合并敏感度、特异度、阳性似然比、阴性似然比及SROC曲线下面积分别为88%、94%、14.56、0.13、0.9736;巢式RT-PCR技术检测的敏感度、特异度及SROC曲线下面积最高,分别为91%、95%、0.9805;普通RT-PCR技术检测的敏感度和特异度次之,分别为87%和94%;定量RT-PCR技术检测的敏感度为80%,特异度为93%。结论:与病理检查相比,RT-PCR技术检测尿液中survivin mRNA诊断膀胱癌有较高的敏感度和特异度,可作为膀胱镜检查的主要辅助手段之一,用于膀胱癌的筛查及术后监测。

RT-PCR/Southern杂交方法检测肝癌患者外周血AFP mRNA

目的 寻找一种敏感的方法以检外外周血中的肝癌细胞。方法 分离原发性肝癌患者以及肝硬化、急慢性肝炎、肝转移癌、肝脏良性肿瘤患者和健康志愿者的外周血单个核细胞 ,提取单个核细胞的总RAN ,进行RT -PCR/Southern杂交检测AFPmRNA。结果 原发性肝癌患者外周血AFPmRAN的检出率为 41.4% ,其检出率与肿瘤大小以及TNM分期有关 ,对照组均为阴性。结论 肝癌患者外周血中的AFPmRNA可以作为肝癌细胞的血液微小转移的标志物。

大鼠TGF-β1 mRNA表达水平的SYBR GreenⅠ荧光定量RT-PCR方法的建立

目的 建立检测大鼠TGF- β1mRNA表达水平的SYBRGreenⅠ荧光定量RT- PCR方法。方法 摸索SYBRGreenⅠ工作液的配制方法,筛选PCR反应液,优化反应中引物和MgCl2 的浓度,以actin为对照,建立大鼠TGF -β1mR NA表达水平的SYBRGreenⅠ荧光定量RT PCR方法并检测大鼠肾皮质TGF -β1mRNA表达水平。结果 SYBRGreenⅠ工作液,4 0倍水溶液稳定性好,最佳反应稀释度为1∶1 0 0 0 0。大鼠TGF β1表达水平的SYBRGreen荧光定量PCR方法,TGF- β1和actin的最佳MgCl2 反应浓度分别为2 5mM和3 5mM ,引物浓度分别为0 . 8μM和0. 5μM ,特异扩增产物的熔解温度分别为88 1和88 .6℃,因此选择在85℃时收集荧光信号。正常大鼠TGF- β1的Ct值在2 9 0 3,actin的Ct值在2 4 .86 ,扩增效率分别为0 . 995和1 . 0 8。结论 通过优化反应条件和特异性分析,成功地建立了特异、敏感的大鼠TGF- β1mRNASYBRGreenⅠ荧光定量RT PCR。SYBRGreenⅠ荧光定量PCR快速、敏感、特异、经济,可以满足临床和科研的需要。

Wilms瘤基因WT_1 mRNA的FQ-RT-PCR定量分析研究

[目的]探讨WT1 mRNA在Wilms瘤表达的临床意义及其与Wilms瘤生物学性状的关系。【方法】对22例Wilms瘤、瘤旁肾组织及血液标本行WT1 mRNA FQ—RT—PCR定量检测。用SPSS软件进行统计学检验,分析。【结果】肿瘤组织中WT1mRNA表达显著高于肾组织和血液,与年龄、性别、BWT或UWT无关。UH 型高于FH型,而肾组织表达又较血液高,BWT血中WT1 mRNA表达高于正常对照组及UWT组,而UWT与正常表达无差别。【结论】Wilms瘤基因WT1 mRNA表达与Wilms瘤发生发展存在关联,且与肿瘤的病理及预后相关.表达越高病理类型及预后越差,但与WT患儿的年龄、性别、BWT或UWT无关。血WT1 mRNA表达水平可能作为BWT的诊断或高危筛选指标。

RT-PCR检测人白细胞钙调素mRNA表达水平与初步应用

目的：检测人白细胞钙调素ｍＲＮＡ表达水平。方法：采用以β２微球蛋白（β２Ｍ）为内标的反转录聚合酶链反应（ＲＴ－ＰＣＲ）技术。结果：建立了人白细胞钙调素ｍＲＮＡ表达水平的半定量测定方法。结论：初步了解不同时期内服用钙拮抗剂（ＣａＡ）的心血管病患者细胞内ＣａＭｍＲＮＡ表达的变化规律。

定量RT-PCR法检测CK20 mRNA、CEAmRNA在胃肠道肿瘤外周血中的表达

目的检测胃癌、大肠癌、健康人外周血CK20 mRNA、CEA mRNA的表达,探讨其临床意义。方法用定量RT-PCR法检测外周血白细胞中CK20mRNA、CEA mRNA的含量,肿瘤组30人,健康对照组23人。结果试验结果显示肿瘤组CEA mRNA、CK20 mRNA表达水平明显高于健康对照组(P=0.000、P=0.000)。分组比较中IV期CK20 mRNA明显高于I～III期,P为0.000,差异显著;IV期与I～III期CEA mRNA没有差别。肿瘤组其中之一阳性28人,阳性率93.75%;两指标同时阳性22人,阳性率73.33%。结论本试验胃癌、大肠癌外周血检测CEA mRNA敏感性和特异性高于CK20 mRNA,但CEA mRNA数值高低与分期无关,CK20 mRNA的表达量与肿瘤分期有关。两指标联合检测未提高阳性率。

Real time RT-PCR定量检测AFP mRNA表达水平方法的建立

目的 建立realtime逆转录聚合酶链反应 (RT PCR)检测AFPmRNA表达水平方法。方法 提取BEL740 2细胞总RNA ,进行RT PCR扩增并纯化AFP基因片段 ,构建重组质粒 ,建立realtimeRT PCR检测AFPmRNA表达水平方法。结果 重组质粒的阳性克隆效率为 81.8% ,经酶切鉴定 ,目的基因片段已插入PMD 18T载体内 ,得到realtimeRT PCR动力学曲线。结论 成功建立了realtimeRT PCR定量检测AFPmRNA表达水平方法。

hMLH1及hPMS2 mRNA在胃癌组织中荧光定量RT-PCR的检测及其临床意义

目的:探讨DNA错配修复基因hMLH1及hPMS2在胃癌组织中的表达水平及其临床意义.方法:应用实时荧光定量逆转录聚合酶链反应(RT-PCR)技术对40例胃癌患者的癌组织、40例癌旁胃炎组织及21例慢性胃炎患者的慢性胃炎组织hMLH1 mRNA及hPMS2 mRNA进行定量检测,以三磷酸甘油醛脱氢酶基因(hGAPDH)为内参照.结果:胃癌组织、癌旁组织、慢性胃炎组织中hMLH1 mRNA的相对含量分别是7.23±11.91,3.80±5.13,2.01±1.25,三组相比差异有统计学意义(F=3.272,均P=0.042),胃癌组织中hMLH1 mRNA含量明显高于其他两组,癌旁组织中含量明显高于慢性胃炎组织中的含量(均P<0.05);各组织中hPMS2 mRNA的相对含量分别是0.43±0.35,0.55±0.39,0.32±0.15,3组相比差异有统计学意义(F=3.349,均P=0.039),胃癌组织与癌旁组织中hPMS2mRNA的含量差异无统计学意义,但均高于慢性胃炎组织中的含量(均P<0.05).除hMLH1 mRNA含量在有、无淋巴结转移的胃癌组织中有显著差异(均P<0.05)外,在胃癌组织中hMLH1 mRNA及hPMS2 mRNA相对含量受肿瘤直径、浸润深度、淋巴结转移的影响不大(均P>0.05).结论:胃癌组织和癌旁组织与慢性胃炎组织相比存在hMLH1及hPMS2 mRNA转录差异,这种基因的转录差异可能与胃癌的发生有关,而与胃癌的发展关系不显著.

实时荧光定量RT-PCR对胃癌组织中hPMS2 mRNA的检测及其临床意义

目的:探讨DNA错配修复基因hPMS2在胃癌组织中的表达水平及其临床意义.方法:应用实时荧光定量逆转录聚合酶链反应(RT-PCR)技术对41例胃癌患者的癌组织、37例癌旁萎缩性胃炎组织、25例慢性萎缩性胃炎组织及20例慢性浅表性胃炎组织中hPMS2 mRNA进行定量检测,以三磷酸甘油醛脱氢酶基因(hGAPDH)为内参照.结果:胃癌组织、癌旁萎缩性胃炎组织、慢性萎缩性胃炎组织及慢性浅表性胃炎组织中hPMS2 mRNA的含量分别是28.33±14.05,16.88±10.67,7.25±8.72和1.78±1.34,四组相比差异有显著性(F=31.82,P=0.00).胃癌组织中hPMS2mRNA含量明显高于其他三组;癌旁萎缩性胃炎组织中含量明显高于非胃癌患者的胃炎组织,差异均有显著性(P<0.01);而非胃癌患者的慢性萎缩性胃炎组织中hPMS2 mRNA的含量与慢性浅表性胃炎组织相比差别无显著性.hPMS2 mRNA含量与肿瘤的大小、浸润深度、有无淋巴结转移关系不明显,差异均无显著性.结论:hPMS2基因的异常转录可能与胃癌的发生有关,而与胃癌的发展关系不大.

The diagnostic significance of the detection of cytokeratin 19 mRNA by quantitative RT-PCR in benign and malignant pleural effusions

Objective: To evaluate the diagnostic significance of detecting cytokeratin 19 (CK19) mRNA by quantitative reverse transcription polymerase chain reaction (RT-PCR) in benign and malignant pleural effusions. Methods: CK19 mRNA was examined by quantitative RT-PCR and CK19 was detected by Enzyme-linked immunoadsorbent assay (ELISA) in 32 patients with malignant pleural effusions and 35 patients with benign pleural effusions. Results: On the threshold of 200 copies/μl, the positive rate of CK19 mRNA in patients with malignant pleural effusions was 62.5%. The positive rates of CK19 mRNA and CK19 in the malignant pleural effusions were significantly higher than those in the benign group (P<0.01). Furthermore, the positive rate of CK19 mRNA was higher than that of CK19 in the malignant group (P<0.05). Conclusion: Detection of CK19 mRNA can be a promising diagnostic marker in differential diagnosis of benign and malignant pleural effusions.

Selection and Evaluation of Optimal Reference Genes for Quantitative Reverse Transcription-Polymerase Chain Reaction Analyses of Gene Expression in Human Spermatozoa

Objective:Optimal reference genes are critical for accurate normalization and reliable interpretation of gene expression quantification data.Recently,several strategies have been utilized for validating reference genes in different human tissues.However,no universal reference genes have been described that accurately summarize transcriptional activity in human spermatozoa.Methods:Using quantitative reverse transcription-polymerase chain reaction(RT-qPCR),we evaluated ten commonly used candidate reference genes between two groups of human cryopreserved donor sperm with different pregnancy rates.We assessed the stability of reference genes using three different algorithms,namely geNorm,NormFinder,and BestKeeper.We then identified the most stable reference genes.Results:Male-enhanced antigen 1(MEA1)was identified as the most stably expressed reference gene,followed by testis-enhanced gene transcript(TEGT).Conclusions:We comprehensively identified MEA1 and TEGT as the most stably expressed reference genes for the normalization of gene expression data in human spermatozoa.

异丹叶大黄素和白藜芦醇对小鼠腹腔巨噬细胞白细胞介素6mRNA表达的影响

目的：观察异丹叶大黄素和白藜芦醇对分别由炎性三肽（ｆＭＬＰ）和钙离子载体Ａ２３１８７（Ａ２３１８７）诱导的小鼠腹腔巨噬细胞白细胞介素６（ｉｎｔｅｒｌｅｕｋｉｎ６，ＩＬ６）ｍＲＮＡ表达水平的影响。方法：用半定量ｍＲＮＡ的ＲＴＰＣＲ。结果：异丹叶大黄素和白藜芦醇在２×１０－６和２×１０－５ｍｏｌ·Ｌ－１浓度下，均能抑制ｆＭＬＰ和Ａ２３１８７诱导的小鼠腹腔巨噬细胞ＩＬ６ｍＲＮＡ的表达。结论：该２个化合物抑制ＩＬ６活性的作用机制之一是抑制ＩＬ６ｍＲＮＡ表达。

降钙素基因相关肽(CGRP)mRNA在大鼠淋巴细胞中的表达(英文)

最近，我们研究发现大鼠胸腺和淋巴结淋巴细胞中存在降钙素基因相关肽（CGRP）样免疫反应活性。应用人工合成的可特异扩增降钙素／降钙素基因相关肽基因部分片断的寡核苷酸引物，通过逆转录-多聚酶链反应（RT－PCR），检测在大鼠脊髓背根神经节、胸腺细胞及肠系膜淋巴结淋巴细胞中是否存在CGRPmRNA，进一步研究大鼠淋巴细胞能否合成CGRP。结果显示，通过RT－PCR从脊髓背根神经节（阳性对照）、胸腺和淋巴结淋巴细胞样品中，都检测到预期的90个碱基对的CGRPCDNA片断。这提示，CGRP除了是一种重要的神经递质外，也可在胸腺细胞和淋巴结淋巴细胞合成。此种CGRP可能与感觉神经源的一样，是免疫和神经系统的共用配基之一。淋巴细胞源的CGRP可能起着免疫细胞调节因子的作用。

胃肠道癌外周血CK20 mRNA的表达及临床意义

背景与目的:检测上皮组织特异性标志物,如CK19、CK20、多形上皮粘蛋白的核心蛋白(MUC1)及CEA等,在外周血、淋巴结及骨髓中的表达,是诊断早期胃肠道癌微转移的重要手段之一。本研究通过检测手术前、后胃肠道癌患者外周血细胞角蛋白20信使核糖核酸(CK20mRNA)的表达,了解胃肠道癌血行播散转移情况及在围手术期综合治疗中的意义。方法:采用巢式逆转录-聚合酶链式反应(Nest-RT-PCR)检测62例胃肠道癌患者手术前后外周血CK20mRNA的表达状况。结果:62例样本中,34例(54.3%)术前CK20mRNA检测为阳性,31例(50.0%)术后CK20mRNA检测为阳性,对照组10例正常人及12例良性胃肠病患者CK20mRNA测定均为阴性。Ⅰ、Ⅱ、Ⅲ、Ⅳ期患者外周血CK20mRNA阳性率依次为37.5%、36.3%、66.7%、100.0%。Ⅰ、Ⅱ期患者与Ⅲ、Ⅳ期患者比较,两者间CK20mRNA的阳性率(分别为36.9%和83.3%)有显著性差异(P<0.05)。胃肠道癌的分化程度及癌栓的形成与外周血CK20mRNA的表达无关。有局部淋巴结转移患者与无淋巴结转移患者的阳性表达率有显著性差异(P<0.01)。手术前后外周血CK20mRNA阳性率差异无显著性(P>0.05)。结论:外周血CK20mRNA检测可作为胃肠道癌微转移的判断指标之一,对临床正确分期及治疗起指导作用。