Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen,Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP,Ecuador)": High quality-low cost local SARS-CoV-2 tests for South America

BackgroundDozens of commercial RT-qPCR kits for SARS-CoV-2 detection are available with or without Emergency Use Authorization (EUA) by FDA or other regulatory agencies.ObjectiveWe evaluated the clinical performance of two SARS-CoV-2 RT-PCR kits designed and produced in South America, "COVID-19 RT-PCR Real TM FAST (CY5)" (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador), for RT-qPCR SARS-CoV2 detection using "TaqMan 2019-nCoV Assay Kit v1" (Thermofisher, USA) as a gold standard technique.ResultsWe report a great clinical performance and analytical sensitivity for the two South American kits with sensitivity values of 96.4 and 100%, specificity of 100% and limit of detection in the range of 10 copies/uL of RNA extraction.Conclusions"COVID-19 RT-PCR Real TM FAST (CY5)" and "ECUGEN SARS-CoV-2 RT-qPCR" kits are reliable SARS-CoV-2 tests made in South America that have been extensively used in Uruguay, Argentina, Brazil, Bolivia and Ecuador. These locally produced SARS-CoV-2 tests have contributed to overcome supply shortages and reduce diagnosis cost, while maintaining the high quality standards of FDA EUA commercially available kits. This approach could be extended for other diagnostic products to improve infectious diseases surveillance at middle and low income countries beyond COVID-19 pandemic.     

RT-qPCR based quantitative analysis of gene expression in single bacterial cells

Recent evidence suggests that cell-to-cell difference at the gene expression level is an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression heterogeneity determines the fate of individual bacterial cells in populations and could also affect the ultimate fate of populations themselves. To quantify the heterogeneity and its biological significance, quantitative methods to measure gene expression in single bacterial cells are needed. In this work, we developed two SYBR Green-based RT-qPCR methods to determine gene expression directly in single bacterial cells. The first method involves a single-tube operation that can analyze one gene from each bacterial cell. The second method is featured by a two-stage protocol that consists of RNA isolation from a single bacterial cell and cDNA synthesis in the first stage, and qPCR in the second stage, which allows determination of expression level of multiple genes simultaneously for single bacterial cells of both gram-positive and negative. We applied the methods to stress-treated (i.e. low pH and high temperature) Escherichia coli populations. The reproducible results demonstrated that the method is sensitive enough not only for measuring cellular responses at the single-cell level, but also for revealing gene expression heterogeneity among the bacterial cells. Furthermore, our results showed that the two-stage method can reproducibly measure multiple highly expressed genes from a single E. coli cell, which exhibits important foundation for future development of a high throughput and lab-on-chips whole-genome RT-qPCR methodology for single bacterial cells.     

Influence of RT-qPCR primer position on EGFR interference efficacy in lung cancer cells

Background  

Real-time quantitative RT-PCR (RT-qPCR) is a "gold" standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets.     

体内转录的多聚腺苷酸加速外源基因mRNA的出核转运

目的:探索体内转录的短多聚腺苷酸[poly(A)]对外源基因mRNA的表达及出核转运的影响。方法:构建依赖于H1启动子体内转录的poly(A)载体,用脂质体转染方法将其与外源绿色荧光蛋白(GFP)基因表达质粒导入MCF-7细胞,采用qPCR和Western印迹分别检测GFP在mRNA和蛋白水平的表达情况,并通过体外实验检测体内转录的poly(A)对GFP mRNA的加尾影响;采用qPCR法考察16种内源基因的mRNA水平;将其与p53表达质粒共转染MCF-7细胞后,采用MTT法检测细胞增殖情况。结果:在MCF-7细胞中,依赖于H1启动子转录的poly(A)能够加速外源GFP mRNA的poly(A)加尾,促进其从细胞核向细胞质输出,从而提高12 h内GFP的表达量,对内源基因mRNA水平没有影响;它还能加速外源p53基因的表达。结论:建立了通过体内转录poly(A)从而加速外源基因表达的策略,可能对基因治疗或快速研究某个特异基因的功能具有重要的应用价值。     

Single-Cell Gene Expression Analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders

Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by single-cell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia, respectively.     

应用激光显微切割技术检测单个结肠细胞的基因表达

目的：探讨检测单个结肠细胞的基因表达的方法。方法：应用激光显微切割技术(1aser micmdissection)从冰冻切片上将单个结肠细胞切下,提取总RNA,将RNA逆转录成cDNA,采用巢式逆转录聚合酶链反应(nested RT—PCR)检测mRNA的表达。结果：在显微镜下用紫外激光显微切割机,将单个结肠细胞成功切下,提取RNA后,逆转录成cDNA,经过巢式RT—PCR扩增后,扩增产物在琼脂糖凝胶上清晰可见。结论：联合应用激光显微切割和巢式RT—PCR可以检测单个结肠细胞的基因表达。     

Comparison of Whole Blood RNA Preservation Tubes and Novel Generation RNA Extraction Kits for Analysis of mRNA and MiRNA Profiles

Background

Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA.

Methods

3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n = 12) and without (n = 35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-)automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n = 72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed.

Results

RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 µg, p<0.001) followed by the choice of blood collection tube. (Semi-)automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed.

Conclusion

We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles.