Global preamplification simplifies targeted mRNA quantification

Kroneis, ThomasGothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för patologi,Sahlgrenska Cancer Center,Institute of Biomedicine, Department of Pathology (author)

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.

Jonasson, Emma,1987Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för patologi,Sahlgrenska Cancer Center,Institute of Biomedicine, Department of Pathology(Swepub:gu)xjemma (author)
Andersson, Daniel,1979Gothenburg University,Göteborgs universitet,Sahlgrenska Cancer Center,Institutionen för biomedicin, avdelningen för patologi,Institute of Biomedicine, Department of Pathology(Swepub:gu)xadanw (author)
Dolatabadi, SoheilaGothenburg University,Göteborgs universitet,Sahlgrenska Cancer Center,Institutionen för biomedicin, avdelningen för patologi,Institute of Biomedicine, Department of Pathology(Swepub:gu)xdolso (author)
Ståhlberg, Anders,1975Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för patologi,Sahlgrenska Cancer Center,Institute of Biomedicine, Department of Pathology(Swepub:gu)xsandw (author)
Göteborgs universitetInstitutionen för biomedicin, avdelningen för patologi (creator_code:org_t)

By the author/editor: Kroneis, Thomas; Jonasson, Emma, ...; Andersson, Danie ...; Dolatabadi, Sohe ...; Ståhlberg, Ander ...

About the subject

MEDICAL AND HEALTH SCIENCES: MEDICAL AND HEAL ...; and Clinical Medicin ...; and Cancer and Oncol ...

MEDICAL AND HEALTH SCIENCES: MEDICAL AND HEAL ...; and Basic Medicine; and Cell and Molecul ...

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