Global preamplification simplifies targeted mRNA quantification.
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.
Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90, Gothenburg, Sweden.|Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, 8010, Graz, Austria.
Thomas
T
Kroneis
Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90, Gothenburg, Sweden.
Emma
E
Jonasson
Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90, Gothenburg, Sweden.
Daniel
D
Andersson
Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90, Gothenburg, Sweden.
Soheila
S
Dolatabadi
Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90, Gothenburg, Sweden.
Anders
A
Ståhlberg
D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods* / Q000592:standards; D059467:Transcriptome
D012333:RNA, Messenger