Autor(es): Fontes, Patricia Kubo [UNESP] ; Castilho, Anthony César Souza [UNESP] ; Razza, Eduardo Montanari [UNESP] ; Nogueira, Marcelo Fábio Gouveia [UNESP]
Data: 2020
Identificador Persistente: http://hdl.handle.net/11449/200080
Origem: Oasisbr
Assunto(s): Gene expression; High-throughput qPCR system; Low RNA concentration; Microfluids; Preamplification; Reproductive system
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Sample types such as those from reproductive systems often yield scarce material, which limits RT-qPCR analysis to only a few targets. Recently developed high-throughput systems can potentially change this scenario, however, the nanoliter scale of such platforms requires extra processing, e.g., preamplification, which needs to be defined through observation and experience. In order to establish best practices in high-throughput PCR approaches using samples from reproductive systems, we evaluated the Biomark™ HD performance using 11 different sample types from the bovine reproductive system: blastocyst (single/pool), oocyte (pool), cumulus, granulosa, and theca cells, oviduct tissue, fetal ovary, testicle (adult/fetal), and uterine horn. We observed that the preamplification step is not just reliable, but mandatory. Our results indicated that 14-preamplification cycles associated to 5- and 7-fold-dilution is the best approach for those samples. Additionally, the Biomark™ HD system has a high intra and inter reproducibility, therefore its performance in duplicate is unnecessary for the ΔCq analysis, taking in consideration the cutoff value 4 < Cq < 22. In summary, this high-throughput approach is a reliable and excellent tool for studying the bovine reproductive system, especially using quantitatively-limited samples, as a larger number of target genes can be assessed from a very low amount of starting material.
Laboratory of Phytomedicines Pharmacology and Biotechnology Department of Pharmacology Institute of Biosciences University of São Paulo State (Unesp) Campus of Botucatu, São Paulo
University of Western São Paulo (Unoeste) Campus of Presidente Prudente, São Paulo
Department of Biological Sciences School of Sciences and Languages São Paulo State University (Unesp) Campus of Assis, São Paulo
Laboratory of Phytomedicines Pharmacology and Biotechnology Department of Pharmacology Institute of Biosciences University of São Paulo State (Unesp) Campus of Botucatu, São Paulo
Department of Biological Sciences School of Sciences and Languages São Paulo State University (Unesp) Campus of Assis, São Paulo
FAPESP: 2012/50533–2
