title: Pre-amplification in the context of high-throughput qPCR gene expression experiment.
abstract: BACKGROUND
  With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm).
  
  
  RESULTS
  For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted.
  
  
  CONCLUSIONS
  We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.
authors.affiliation: Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. vlasta.korenkova@ibt.cas.cz.
authors.forename: Vlasta
authors.initials: V
authors.lastname: Korenková
authors.affiliation: QFAB Bioinformatics, University of Queensland - St Lucia QLD, Brisbane, Australia. j.scott@qfab.org.
authors.forename: Justin
authors.initials: J
authors.lastname: Scott
authors.affiliation: Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. vendula.novosadova@ibt.cas.cz.
authors.forename: Vendula
authors.initials: V
authors.lastname: Novosadová
authors.affiliation: Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. M.Jindrichova@seznam.cz.
authors.forename: Marie
authors.initials: M
authors.lastname: Jindřichová
authors.affiliation: Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. lucie.langerova@ibt.cas.cz.
authors.forename: Lucie
authors.initials: L
authors.lastname: Langerová
authors.affiliation: Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. david.svec@ibt.cas.cz.
authors.forename: David
authors.initials: D
authors.lastname: Švec
authors.affiliation: Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. monika.sidova@ibt.cas.cz.
authors.forename: Monika
authors.initials: M
authors.lastname: Šídová
authors.affiliation: TATAA Biocenter, Göthenburg, Sweden. robert.sjoback@tataa.com.
authors.forename: Robert
authors.initials: R
authors.lastname: Sjöback
mesh_terms: D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods; D064368:Healthy Volunteers; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis / Q000097:blood / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D012333:RNA, Messenger