کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7232248 | 1470962 | 2015 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10 µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350 µm at a printing distance of 6 mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biosensors and Bioelectronics - Volume 69, 15 July 2015, Pages 301-306
Journal: Biosensors and Bioelectronics - Volume 69, 15 July 2015, Pages 301-306
نویسندگان
F. Stumpf, J. Schoendube, A. Gross, C. Rath, S. Niekrawietz, P. Koltay, G. Roth,