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Pre-amplification in the context of high-throughput qPCR gene expression experiment
V. Korenková, J. Scott, V. Novosadová, M. Jindřichová, L. Langerová, D. Švec, M. Šídová, R. Sjöback,
Jazyk angličtina Země Anglie, Velká Británie
Typ dokumentu časopisecké články, práce podpořená grantem
NLK
BioMedCentral
od 2000-12-01 do 2019-12-31
BioMedCentral Open Access
od 2000
Free Medical Journals
od 2000
PubMed Central
od 2000 do 2019
Europe PubMed Central
od 2000
ProQuest Central
od 2009-01-01 do 2019-01-31
Open Access Digital Library
od 2000-10-01
Open Access Digital Library
od 2000-01-01
Open Access Digital Library
od 2000-01-01
Medline Complete (EBSCOhost)
od 2000-01-01 do 2019-11-08
Health & Medicine (ProQuest)
od 2009-01-01 do 2019-01-31
Springer Nature OA/Free Journals
od 2000-12-01 do 2019-12-31
- MeSH
- lidé MeSH
- messenger RNA analýza krev metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí přístrojové vybavení metody MeSH
- senzitivita a specificita MeSH
- stanovení celkové genové exprese přístrojové vybavení metody MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
Citace poskytuje Crossref.org
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- $a 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.
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