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Thoracic muscle tissue was removed from large specimens for extraction.
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To ensure that the results are maximally informative to allow retrospective genetic analyses, we have used a consistent set of historical specimens for all extraction methods, and assessed the quality of the nucleic acids using up to 10 different assays.
Clinicopathological information and biologic specimens for DNA extraction were obtained from all participating families with the informed consent of the mothers and fathers, and all aspects of this research were in compliance with the tenets of the Declaration of Helsinki for human research (http://www.wma.net).
Clinicopathological information from all participating families and biologic specimens for DNA extraction were obtained with the written informed consent of the mothers and fathers, and all aspects of this research were in compliance with the tenets of the Declaration of Helsinki for human research (http://www.wma.net).
All wild-type cases with sufficient residual FFPE specimens for RNA extraction were included in this study.
Routine handling of clinical specimens for RNA extraction and serologic testing by immunofluorescence were done in a BSL-2 laboratory.
Legs were removed from adults of the larger species, Ptychoptera, Bittacomorphella, Paracladura, Cramptonomyia, and Sylvicola specimens for separate extraction.
Specimens for DNA extraction were chosen to represent samples from different collection sites and, in the case of G. sagittariae, to represent different host plant preferences.
Specimens for RNA extraction and gene expression profile analysis were stored in RNA later (Sigma-Aldrich, Milano, Italy) at 4° C for 24 h and then snap-frozen in liquid nitrogen and stored at -80° C until use.
Single specimens were macerated for extraction, only specimens of Ctenocephalides felis were pooled.
These two specimens were pooled for extraction of RNA.
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