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Using the collected tissues and cell populations, cDNA libraries were constructed by one of the following three methods.
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RNA was extracted from the collected tissues using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed to cDNA using Superscript III (Invitrogen).
Total RNA was isolated from the collected tissues (e.g. brain) using the Illustra RNAspin minikit from GE Healthcare (Little Chalfont, UK) with on-column DNase I treatment.
In all cases, patients gave separate written consent for the use of the collected tissue.
The collected tissue samples were used without further processing.
Retrospective research using already collected tissue and data obtains an ethics waiver, thereby avoiding the requirement of re-consent, or it re-contacts and re-consents participants where feasible, or, finally, it anonymizes the data and samples, thereby limiting their usefulness to meta-analyses or as controls [ 37].
All necropsy submissions to the VMDL include permission for use of collected tissues in future research.
Written informed consent was obtained from all patients for subsequent use of their collected tissues.
Wild type M. truncatula plants and Nicotiana tabacum (tobacco) Tnt1 retrotransposon tagged Medicago mutants [ 47] were grown in the glasshouse, and used for collecting tissues for molecular and chemical characterization.
Shown are the unique frog IDs and the dates of capture as well as the methods used to collect tissue samples for the PCR assay (BR = bag rinse, TC = toe clip, SW = swab).
JC has provided the mice used to collect tissue samples.
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