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. 2022 Jan 22;23(3):1226. doi: 10.3390/ijms23031226

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© 2022 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Study design. (A) Four library preparation protocols were selected in order to evaluate their performance and usability. Six technical replicates from a pool of plasma samples and aliquots of miRXplore Universal Reference were analysed. (B) The NGS assay utilizing mind spike-ins was designed using the following steps: (1) RNA extraction; (2) adding of the miND spike-in—a pool of 7 oligonucleotides, each containing a 13 nt core sequence flanked by a set of 4 randomized nucleotides on the 5′ and 3′ ends, that were mixed in the defined ratio; (3) preparation of the NGS libraries according to the selected protocols; (4) data analyses and data normalisation based on the miND spike-in concentration range.