Evaluation and Standardization of RNA Extractions with Quality for RNA-Seq for Balamuthia mandrillaris

Parasitologia Pub Date : 2024-06-09 DOI:10.3390/parasitologia4020017

Leobardo Daniel Gonzalez-Zuñiga, L. Z. Rodriguez-Anaya, J. R. Gonzalez-Galaviz, Abraham Cruz-Mendívil, F. Lares-Villa, L. F. Lares-Jiménez

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Abstract

Balamuthia mandrillaris is a free-living amoeba (FLA) that causes granulomatous amebic encephalitis (GAE) and skin lesions. Transcriptomic analysis is a powerful tool used to study B. mandrillaris pathogenic infections. However, preliminary tests of RNA extraction showed poor results, so it has become essential to standardize a protocol for high-quality RNA. The present study evaluated 11 RNA extraction protocols based on three commercial kits by making modifications to the temperature and centrifugation times, and by combining kits. Four protocols, namely Q3 (based on QIAGEN RNeasy Mini Kit, with modifications in temperature and centrifugation times), T1 (Invitrogen TRIzol Reagent), T2 (combination of TRIzol and QIAGEN modified protocols) and T3 (combination of TRIzol and PROMEGA SV Total RNA Isolation protocols), presented RNA with good integrity and purity, except for the T1 protocol, which obtained an A260/230 value below the acceptable threshold. High RNA integrity (RIN) values were obtained with the Q3 (9.8), T2 (9.2), and T3 (8.9) protocols, while the T1 protocol obtained a lower RIN value (7.1). The Q3, T2, and T3 protocols obtained high-quality RNA from B. mandrillaris based on the criteria of integrity, purity, and concentration, where the implemented modifications and combinations raised the quality; thus, their use is recommended to obtain accurate results when performing transcriptomic analysis.

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对山魈RNA-Seq质量的RNA提取物进行评估和标准化

山魈巴拉穆氏阿米巴（Balamuthia mandrillaris）是一种自由生活阿米巴（FLA），可引起肉芽肿阿米巴脑炎（GAE）和皮肤病变。转录组分析是研究山德拉氏阿米巴虫致病性感染的有力工具。然而，RNA 提取的初步测试结果显示效果不佳，因此有必要对高质量 RNA 的提取方案进行标准化。本研究通过修改温度和离心时间以及组合试剂盒，对基于三种商业试剂盒的 11 种 RNA 提取方案进行了评估。除 T1 提取方案的 A260/230 值低于可接受的阈值外，其他四种方案，即 Q3（基于 QIAGEN RNeasy Mini Kit，修改了温度和离心时间）、T1（Invitrogen TRIzol Reagent）、T2（TRIzol 和 QIAGEN 修改方案的组合）和 T3（TRIzol 和 PROMEGA SV 总 RNA 分离方案的组合），均能获得完整性和纯度良好的 RNA。Q3 （9.8）、T2（9.2）和 T3（8.9）方案获得了较高的 RNA 完整性（RIN）值，而 T1 方案获得了较低的 RIN 值（7.1）。根据完整性、纯度和浓度标准，Q3、T2 和 T3 方案从山魈中获得了高质量的 RNA，其中实施的修改和组合提高了质量；因此，建议在进行转录组分析时使用这些方案以获得准确的结果。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Parasitologia

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