Title:	Optimization of genomic DNA and total RNA extraction protocols from microlepidopterans for high-fidelity long-read sequencing.
Authors:	NASCIMENTO, E. F. M. B. QUEIROZ, E. L. S. VIDAL, L. A. JUNQUEIRA, C. I. C. V. F. SOUZA, B. H. S. MANIGAT, D. MARANHÃO, A. Q. FREIRE, E. V. S. A.
Affiliation:	ELIZA F. M. B. NASCIMENTO; ERICK L. S. QUEIROZ, UNIVERSITY OF BRASÍLIA; LEONARDO A. VIDAL; CAMILA I. C. V. F. JUNQUEIRA; BRUNO H. S. SOUZA, FEDERAL UNIVERSITY OF LAVRAS; DONALD MANIGAT, FEDERAL UNIVERSITY OF AMAPÁ; ANDREA Q. MARANHÃO, UNIVERSITY OF BRASÍLIA; ERIKA VALERIA SALIBA ALBUQUERQUE FREIRE, CENARGEN.
Date Issued:	2025
Citation:	BMC Methods, v. 2, 19 2025.
Description:	Background Microlepidoptera, for the most part, are important agricultural pests capable of causing economic losses in different global crops, highlighting the need for biotechnological control strategies. They are small species, with a cryptic behaviors and high chitin content in their structure. Furthermore, they have eating habits that can make it difficult to extract high-quality gDNA and RNA. The limited number of existing protocols to obtain high mass nucleic acids from smaller insects render difficult to obtain elevated purity and integrity standards. We present straightforward and reproducible protocol modifications for extracting DNA and RNA for PacBio and Illumina sequencing from Leucoptera coffeella, also validated in Plutella xylostella and Tuta absoluta. Methods Genomic DNA was extracted from a pool of 20 L. coffeella pupae using a modified protocol based on a commercial extraction kit, performed over two days. Key optimizations included cell lysis with CTL buffer, use of wide-bore pipette tips, extended and agitated incubation during protein digestion, and elution at room temperature with a reduced buffer volume. Total RNA was extracted from all seven developmental stages of L. coffeella in 2 h and 35 min. In addition to standard protocol steps, an extra purification step using a commercial kit was included to improve RNA quality. Discussion Compared to other extraction methods, these protocols showed higher degree purity and concentration. The extracted samples presented concentrations, absorbance and integrity relationships recommended and validated by HiFi sequencing. The extraction of nucleic acids from microlepidopterans may present several challenges, such as the small size and fragility of the samples and contamination by microorganisms, which can affect the quality of the genetic material obtained. The optimization described here provides significant improvements for obtaining gDNA and RNA from L. coffeella and can be applied to other insect species, particularly microlepidoptera, as here validated in P. xylostella and T. absoluta. Essential for genomic and transcriptomic studies and also enhance various molecular techniques, such as PCR, qPCR, molecular markers, and others.
Keywords:	Genetic material L coffeella Illumina PacBio
DOI:	https://doi.org/10.1186/s44330-025-00038-3
Notes:	Na publicação: Érika V. S. Albuquerque.
Type of Material:	Artigo de periódico
Access:	openAccess
Appears in Collections:	Artigo em periódico indexado (CENARGEN)