High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology.
Journal of visualized experiments : JoVE
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Chauve, L., Le Pen, J., Hodge, F., Todtenhaupt, P., Biggins, L., Miska, E., Andrews, S. R., & et al. (2020). High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology.. Journal of visualized experiments : JoVE, (159)https://doi.org/10.3791/61132
This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named 'Worm-to-CT', allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.
Animals, Caenorhabditis elegans, Heat-Shock Proteins, Caenorhabditis elegans Proteins, DNA, Helminth, DNA, Complementary, RNA, Messenger, DNA Primers, Oligonucleotide Array Sequence Analysis, Reverse Transcriptase Polymerase Chain Reaction, Real-Time Polymerase Chain Reaction
WELLCOME TRUST (104640/Z/14/Z)
Wellcome Trust (093970/Z/10/Z)
External DOI: https://doi.org/10.3791/61132
This record's URL: https://www.repository.cam.ac.uk/handle/1810/307097
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/