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

Overview of attention for article published in BMC Molecular and Cell Biology, March 2015
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About this Attention Score

  • In the top 25% of all research outputs scored by Altmetric
  • Among the highest-scoring outputs from this source (#39 of 1,233)
  • High Attention Score compared to outputs of the same age (85th percentile)
  • High Attention Score compared to outputs of the same age and source (89th percentile)

Mentioned by

blogs
1 blog
patent
2 patents

Citations

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37 Dimensions

Readers on

mendeley
127 Mendeley
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Title
Pre-amplification in the context of high-throughput qPCR gene expression experiment
Published in
BMC Molecular and Cell Biology, March 2015
DOI 10.1186/s12867-015-0033-9
Pubmed ID
Authors

Vlasta Korenková, Justin Scott, Vendula Novosadová, Marie Jindřichová, Lucie Langerová, David Švec, Monika Šídová, Robert Sjöback

Abstract

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). 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. 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.

Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 127 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
United States 1 <1%
Denmark 1 <1%
Canada 1 <1%
Unknown 124 98%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 30 24%
Researcher 29 23%
Student > Master 17 13%
Student > Bachelor 11 9%
Other 8 6%
Other 11 9%
Unknown 21 17%
Readers by discipline Count As %
Agricultural and Biological Sciences 40 31%
Biochemistry, Genetics and Molecular Biology 37 29%
Engineering 7 6%
Immunology and Microbiology 5 4%
Neuroscience 4 3%
Other 12 9%
Unknown 22 17%
Attention Score in Context

Attention Score in Context

This research output has an Altmetric Attention Score of 11. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 20 June 2022.
All research outputs
#3,138,412
of 25,373,627 outputs
Outputs from BMC Molecular and Cell Biology
#39
of 1,233 outputs
Outputs of similar age
#38,217
of 274,513 outputs
Outputs of similar age from BMC Molecular and Cell Biology
#2
of 19 outputs
Altmetric has tracked 25,373,627 research outputs across all sources so far. Compared to these this one has done well and is in the 87th percentile: it's in the top 25% of all research outputs ever tracked by Altmetric.
So far Altmetric has tracked 1,233 research outputs from this source. They receive a mean Attention Score of 4.0. This one has done particularly well, scoring higher than 96% of its peers.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 274,513 tracked outputs that were published within six weeks on either side of this one in any source. This one has done well, scoring higher than 85% of its contemporaries.
We're also able to compare this research output to 19 others from the same source and published within six weeks on either side of this one. This one has done well, scoring higher than 89% of its contemporaries.