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Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

Overview of attention for article published in BMC Research Notes, November 2017
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  • Above-average Attention Score compared to outputs of the same age (51st percentile)
  • Above-average Attention Score compared to outputs of the same age and source (61st percentile)

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3 tweeters

Citations

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33 Mendeley
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Title
Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines
Published in
BMC Research Notes, November 2017
DOI 10.1186/s13104-017-2959-0
Pubmed ID
Authors

Roslyn L. Mainland, Taylor A. Lyons, Mike M. Ruth, Jamie M. Kramer

Abstract

RNA interference is employed extensively in Drosophila research to study gene function within a specific cell-type or tissue. Thousands of transgenic Drosophila lines have been generated to express double stranded RNA for gene knockdown; however, no standardized method exists for quantifying their knockdown efficiency. Since antibodies are not available for many proteins, quantitative real-time PCR is often used. Here, we explore how primer design and RNA isolation method can influence detection of gene knockdown using qPCR. We tested differences in detected gene knockdown efficiency when using purified polyadenylated mRNA or total RNA as templates for cDNA synthesis. We also tested two different primer locations for each gene: one to amplify a region 5' of the RNAi cut site, and one to amplify a region 3' of the cut site. Consistently, the strongest gene knockdown was detected when qPCR was performed using 5' primer sets in combination with mRNA-derived cDNA. Our results indicate that detection of undegraded mRNA cleavage fragments can result in underestimation of true knockdown efficiency for a RNAi construct. Purification of polyadenylated mRNA, combined with primers designed to amplify the non-polyadenylated 5' mRNA cleavage fragment can avoid this problem.

Twitter Demographics

The data shown below were collected from the profiles of 3 tweeters who shared this research output. Click here to find out more about how the information was compiled.

Mendeley readers

The data shown below were compiled from readership statistics for 33 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 33 100%

Demographic breakdown

Readers by professional status Count As %
Student > Master 10 30%
Student > Ph. D. Student 7 21%
Student > Bachelor 4 12%
Researcher 3 9%
Student > Doctoral Student 2 6%
Other 2 6%
Unknown 5 15%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 12 36%
Agricultural and Biological Sciences 8 24%
Neuroscience 4 12%
Medicine and Dentistry 2 6%
Social Sciences 1 3%
Other 1 3%
Unknown 5 15%

Attention Score in Context

This research output has an Altmetric Attention Score of 2. 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 25 June 2019.
All research outputs
#8,784,870
of 15,323,046 outputs
Outputs from BMC Research Notes
#1,353
of 3,393 outputs
Outputs of similar age
#191,093
of 407,916 outputs
Outputs of similar age from BMC Research Notes
#145
of 418 outputs
Altmetric has tracked 15,323,046 research outputs across all sources so far. This one is in the 41st percentile – i.e., 41% of other outputs scored the same or lower than it.
So far Altmetric has tracked 3,393 research outputs from this source. They receive a mean Attention Score of 4.5. This one has gotten more attention than average, scoring higher than 58% 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 407,916 tracked outputs that were published within six weeks on either side of this one in any source. This one has gotten more attention than average, scoring higher than 51% of its contemporaries.
We're also able to compare this research output to 418 others from the same source and published within six weeks on either side of this one. This one has gotten more attention than average, scoring higher than 61% of its contemporaries.