Effect of DNase treatment on RNA extraction from preimplantation murine embryos

Abstract

ABSTRACT. The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNase- treated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control.

An ultrafast SARS-CoV-2 virus enrichment and extraction method compatible with multiple modalities for RNA detection

“…43 , 44 The DNase and associated buffer were added as part of the rnaGEM extraction cocktail; no additional steps were added to the extraction workflow. Although DNase treatments have been shown to decrease RNA yield, 45 , 46 parallel extractions from clinical samples with and without DNase suggest that its inclusion was not detrimental to SARS-CoV-2 detection ( Fig. S2 ).…”

Urinary extracellular vesicles: Assessment of pre‐analytical variables and development of a quality control with focus on transcriptomic biomarker research

“…However, the amount of DNA is generally low in cell‐free samples, including urine (Oreskovic et al., 2019 ; Streleckiene et al., 2018 ). As the RNA content of EV samples is also low in comparison to cell or tissue RNA and the DNAse treatment reduces RNA yields (Norhazlin et al., 2015 ; Pang et al., 2020 ), the risk of losing the precious EV‐RNA due to the treatment is high. We thus regarded relevant to investigate the necessity of the DNAse treatment by analysing RNA yield and mRNA‐seq results from uEV.…”

Optimization of sperm RNA processing for developmental research

“…Generally, DNase is used to rule out gDNA contamination in isolated RNA. However, DNase has harmful effect not only in gDNA but also in concentrations of RNA and small RNAs 57 , 58 . In this study, DNase affected RNA concentration and quality in RNAlater-treated samples.…”