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.

Subject headings

MEDICIN OCH HÄLSOVETENSKAP  -- Klinisk medicin -- Cancer och onkologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Clinical Medicine -- Cancer and Oncology (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)

Keyword

real-time pcr

single-cell

myxoid liposarcomas

seq

dna

heterogeneity

amplification

smart-seq2

separation

capture

Science & Technology - Other Topics

Publication and Content Type

ref (subject category)

art (subject category)