Pre-amplification in the context of high-throughput qPCR gene expression experiment

Korenková, Vlasta, Scott, Justin, Novosadová, Vendula, Jindřichová, Marie, Langerová, Lucie, Švec, David, Šídová, Monika and Sjöback, Robert (2015) Pre-amplification in the context of high-throughput qPCR gene expression experiment. BMC Molecular Biology, 16 1-10. doi:10.1186/s12867-015-0033-9

Author Korenková, Vlasta
Scott, Justin
Novosadová, Vendula
Jindřichová, Marie
Langerová, Lucie
Švec, David
Šídová, Monika
Sjöback, Robert
Title Pre-amplification in the context of high-throughput qPCR gene expression experiment
Journal name BMC Molecular Biology   Check publisher's open access policy
ISSN 1471-2199
Publication date 2015-03-11
Year available 2015
Sub-type Article (original research)
DOI 10.1186/s12867-015-0033-9
Open Access Status DOI
Volume 16
Start page 1
End page 10
Total pages 10
Place of publication London, United Kingdom
Publisher BioMed Central
Collection year 2016
Language eng
Formatted 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.
Keyword High-throughput qPCR
Exponential pre-amplification
Gene expression
Degraded samples
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
Institute for Molecular Bioscience - Publications
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Citation counts: TR Web of Science Citation Count  Cited 2 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 3 times in Scopus Article | Citations
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