Highly multiplexed single-cell quantitative PCR
| العنوان: | Highly multiplexed single-cell quantitative PCR |
|---|---|
| المؤلفون: | Michael VanInsberghe, Adam K. White, Carl L. Hansen, Hans Zahn, Oleh Petriv |
| المصدر: | PLoS ONE PLoS ONE, Vol 13, Iss 1, p e0191601 (2018) |
| بيانات النشر: | Public Library of Science, 2018. |
| سنة النشر: | 2018 |
| مصطلحات موضوعية: | 0301 basic medicine, Imaging Techniques, Nucleic acid synthesis, Microfluidics, lcsh:Medicine, cDNA synthesis, Genomics, Artificial Gene Amplification and Extension, Computational biology, Research and Analysis Methods, Biochemistry, Polymerase Chain Reaction, 03 medical and health sciences, 0302 clinical medicine, Limit of Detection, Complementary DNA, microRNA, Multiplex polymerase chain reaction, Fluorescence Imaging, Genetics, Chemical synthesis, lcsh:Science, Non-coding RNA, Molecular Biology Techniques, Gene, Molecular Biology, Multidisciplinary, Biology and life sciences, Chemistry, lcsh:R, RNA, Reverse Transcription, Gene regulation, Gene expression profiling, Nucleic acids, MicroRNAs, Biosynthetic techniques, 030104 developmental biology, Real-time polymerase chain reaction, Engineering and Technology, lcsh:Q, Gene expression, Fluidics, Multiplex Polymerase Chain Reaction, 030217 neurology & neurosurgery, Research Article |
| الوصف: | We present a microfluidic device for rapid gene expression profiling in single cells using multiplexed quantitative polymerase chain reaction (qPCR). This device integrates all processing steps, including cell isolation and lysis, complementary DNA synthesis, pre-amplification, sample splitting, and measurement in twenty separate qPCR reactions. Each of these steps is performed in parallel on up to 200 single cells per run. Experiments performed on dilutions of purified RNA establish assay linearity over a dynamic range of at least 104, a qPCR precision of 15%, and detection sensitivity down to a single cDNA molecule. We demonstrate the application of our device for rapid profiling of microRNA expression in single cells. Measurements performed on a panel of twenty miRNAs in two types of cells revealed clear cell-to-cell heterogeneity, with evidence of spontaneous differentiation manifested as distinct expression signatures. Highly multiplexed microfluidic RT-qPCR fills a gap in current capabilities for single-cell analysis, providing a rapid and cost-effective approach for profiling panels of marker genes, thereby complementing single-cell genomics methods that are best suited for global analysis and discovery. We expect this approach to enable new studies requiring fast, cost-effective, and precise measurements across hundreds of single cells. |
| اللغة: | English |
| تدمد: | 1932-6203 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6d65a83d77e69329abc77eeefa4ca6e3Test http://europepmc.org/articles/PMC5788347Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....6d65a83d77e69329abc77eeefa4ca6e3 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 19326203 |
|---|