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Global preamplification simplifies targeted mRNA quantification

Journal article
Authors Thomas Kroneis
Emma Jonasson
Daniel Andersson
Soheila Dolatabadi
Anders Ståhlberg
Published in Scientific Reports
Volume 7
ISSN 2045-2322
Publication year 2017
Published at Institute of Biomedicine, Department of Pathology
Sahlgrenska Cancer Center
Language en
Keywords real-time pcr, single-cell, myxoid liposarcomas, seq, dna, heterogeneity, amplification, smart-seq2, separation, capture, Science & Technology - Other Topics
Subject categories Pathology, Cancer and Oncology


The need to perform gene expression profiling using next generation sequencing and quantitative real-time PCR (qPCR) on small sample sizes and single cells is rapidly expanding. However, to analyse few molecules, preamplification is required. Here, we studied global and target-specific preamplification using 96 optimised qPCR assays. To evaluate the preamplification strategies, we monitored the reactions in real-time using SYBR Green I detection chemistry followed by melting curve analysis. Next, we compared yield and reproducibility of global preamplification to that of target-specific preamplification by qPCR using the same amount of total RNA. Global preamplification generated 9.3-fold lower yield and 1.6-fold lower reproducibility than target-specific preamplification. However, the performance of global preamplification is sufficient for most downstream applications and offers several advantages over target-specific preamplification. To demonstrate the potential of global preamplification we analysed the expression of 15 genes in 60 single cells. In conclusion, we show that global preamplification simplifies targeted gene expression profiling of small sample sizes by a flexible workflow. We outline the pros and cons for global preamplification compared to target-specific preamplification.

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