title: Properties of targeted preamplification in DNA and cDNA quantification.
abstract: OBJECTIVE
  Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR).
  
  
  METHODS
  To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR.
  
  
  RESULTS
  The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification.
  
  
  CONCLUSIONS
  On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.
authors.affiliation: Sahlgrenska Cancer Center, Department of Pathology, Sahlgrenska Academy at University of Gothenburg, Box 425, 40530 Gothenburg, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Andersson
authors.forename: Nina
authors.initials: N
authors.lastname: Akrap
authors.forename: David
authors.initials: D
authors.lastname: Svec
authors.forename: Tony E
authors.initials: TE
authors.lastname: Godfrey
authors.forename: Mikael
authors.initials: M
authors.lastname: Kubista
authors.forename: Göran
authors.initials: G
authors.lastname: Landberg
authors.forename: Anders
authors.initials: A
authors.lastname: Ståhlberg
mesh_terms: D045744:Cell Line, Tumor; D004247:DNA / Q000032:analysis* / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D018076:DNA, Complementary; D004247:DNA
keywords: experimental design; multiplex PCR; preamplification; primer-pools; quantitative real-time PCR; single-cell analysis; targeted preamplification