BioTechniquesVol. 50, No. 1 BioSpotlightOpen AccessBioSpotlightNathan S. Blow & Patrick C.H. LoNathan S. BlowSearch for more papers by this author & Patrick C.H. LoSearch for more papers by this authorPublished Online:28 Jun 2018https://doi.org/10.2144/000113580AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Automatic for the PeopleWhen it comes to studying gene or microRNA expression patterns, quantitative PCR (qPCR) is still the method of choice. While in a low-throughput format the equipment to perform qPCR and the downstream analysis tools are readily available, when it comes to higher-throughput applications and studies, automating the assay can be challenging. One of the major stumbling blocks comes from the rigid robotic scripts that are often supplied with liquid handling robots, making optimization for complicated applications such as qPCR intricate and time-consuming. In the current issue of BioTechniques, S. Callejas and colleagues from the Centro Nacional de Investigaciones Cardiovasculares in Madrid describe a new program they developed called Automatic Genomics which can be used for designing medium-throughput qPCR experiments and load 96- and 384-well plates. The software is based on Microsoft Excel with a Visual Basic interface and requires only a limited programming background to execute. Subapplications give users the opportunity to automate steps such as sample transfer, reverse transcription, and volume calculations according to their defined parameters without the need for specific vendor-supplied scripts. To validate their software, the authors tested technical reproducibility for three genes with 88 cDNA samples, with each sample-gene combination loaded in triplicate. The final data showed that 96% of the sample-gene combinations passed the technical quality control metric cutoff of 0.5 cycles (the maximum allowable difference in cycles between the technical replicates) and 61.5% passed a cutoff of 0.2 cycles. The authors note that this level of reproducibility is higher than what could have been achieved by hand. Although designed for use specifically with the Freedom EVO series of liquid handling robots, this software will enable researchers using those platforms to more easily perform larger scale qPCR experiments, with high reproducibility, without the need for complicated modifications to robotics scripts for each experiment.See “Automatic Genomics: a user-friendly program for the automatic designing and plate loading of medium-throughput qPCR experiments”Convection, ‘Vection, What's Your Function?Continuous improvements in PCR thermocycler designs have significantly reduced instrument size, cost, complexity, and amplification times, facilitating the development of simplified platforms suitable for point-of-care applications. One alternative to traditional PCR is convective PCR, where the sample is repeatedly circulated by convection through different temperature zones corresponding to the denaturation, annealing, and extension steps. Previous convective PCR systems, however, have relied on sophisticated temperature controllers and complex designs requiring tubing and chambers to circulate samples, which has limited their widespread usage. In this issue, P.J. Chen and colleagues at the National Taiwan University (Taipei, Taiwan) describe a greatly simplified convective PCR platform that dispenses with these requirements by carrying out amplification in a single capillary tube placed on a dry heat bath set at 95°C. In their capillary convective PCR (CCPCR) approach, the heated base acts to both denature the DNA sample at the bottom of the tube and also cause it to rise by convection. As this portion of the sample rises, it is cooled by the air surrounding the upper part of the tube and undergoes the annealing and extension steps of PCR, after which it sinks down to begin another cycle of convective PCR. Complete amplification can be achieved in as little as 30–40 minutes and does not require any of the complicated manipulations necessary with previous convective PCR systems. The authors demonstrate that CCPCR can amplify fragments up to 500 bp starting from as few as 30 copies, although proper design of amplicons and primers is required such that their Td and Tm, respectively, are optimized for convective PCR. The hardware requirements of the CCPCR platform can be further reduced by using a basic water bath or the heating plate contained in a portable scent-based mosquito repellent. The low cost and ease of operation of CCPCR makes it ideally suited for use in less-developed countries for the rapid detection of disease outbreaks and point-of-care applications.Schematic diagram of CCPCR illustrating the locations of the different steps of PCR along the temperature gradient of the capillary tube heated at 95°C from the bottom.See “Rapid amplification in a capillary tube by natural convection with a single isothermal heater”FiguresReferencesRelatedDetails Vol. 50, No. 1 Follow us on social media for the latest updates Metrics History Published online 28 June 2018 Published in print January 2011 Information© 2011 Author(s)PDF download
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Mar 1, 2014
Christopher D Bahl + 7
Open Access
