Bona fide gene expression analysis of samples from the bovine reproductive system by microfluidic platform

Autor(es)

Fontes, Patricia Kubo ^[1] ; Souza Castilho, Anthony Cesar ^{[1, 2]} ; Razza, Eduardo Montanari ^[1] ; Gouveia Nogueira, Marcelo Fabio ^{[1, 3]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo State Unesp, Dept Pharmacol, Lab Phytomed Pharmacol & Biotechnol, Campus Botucatu, Sao Paulo - Brazil

^[2] Univ Western Sao Paulo Unoeste, Campus Presidente Prudente, Sao Paulo - Brazil

^[3] Sao Paulo State Univ Unesp, Sch Sci & Languages, Dept Biol Sci, Campus Assis, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Analytical Biochemistry; v. 596, MAY 1 2020.
• Citações Web of Science: 0

Resumo

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 (TM) 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 (TM) HD system has a high intra and inter reproducibility, therefore its performance in duplicate is unnecessary for the Delta Cq 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. (AU)

• Processo FAPESP: 12/50533-2 - GIFT: melhoramento genômico de características relacionadas com a fertilização em gado bovino dinamarquês e brasileiro
• Beneficiário: Marcelo Fábio Gouveia Nogueira
• Modalidade de apoio: Auxílio à Pesquisa - Temático