Comparison of phenol-based and alternative RNA isolation methods for gene expression analyses

Jakovljevic, Ksenija; Spasić, Milena R.; Mališić, Emina; Dobričić, Jelena; Krivokuća, Ana; Janković, Radmila

doi:10.2298/jsc091223084j

Cited by 4 publications

(5 citation statements)

References 6 publications

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“…RNA quality is usually judged in three ways. Firstly, agarose gel electrophoresis demonstrated intact and bright bands representing the ribosomal subunits of the undegraded total RNA [20,[56][57][58]. Secondly, measurement of absorbance 1-The absorption ratios A260/230 and A260/280 were used to detect polysaccharide/polyphenolic contaminants, protein contaminants and high salt, respectively [33,59].…”

Section: Resultsmentioning

confidence: 99%

High RNA quality extracted from the tolerant crop Cyamopsis tetragonoloba (L.) despite possession of low RNA integrity number

Gaafar

Al‐Qurainy

Alshameri

et al. 2021

Biotechnology & Biotechnological Equipment

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Salinity is one of the most limiting constraints of crop production. Nevertheless, highly-tolerant crop plants (e.g. 'Guar' Cyamopsis tetragonoloba) can thrive well under salinity conditions. RNA-Seq plays a central role in identifying genes involved in the response mechanisms to salt stress in plant species. High-quality RNA, free of genomic DNA is a critical determinant for success in subsequent downstream experiments to shed light on tolerance mechanisms. In this research, an inspection of two different total RNA isolation protocols was employed for preparing highly pure and intact RNAs with good yield from Guar tissue under both high salinity and control conditions. The extracted RNA appeared in all quality metrics to be of adequate quantity and quality for most downstream applications, including high-throughput transcriptome sequencing and expression analysis. However, the outcomes showed that the most widely used RNA quality measurement, the RNA Integrity Number (RIN), is a poor indicator with a weak correlation to the integrity of RNA from this plant. In contrast to human and animal cells, plants have various ribosomal RNA species. Consequently, whenever the RNA was separated on an agarose gel, several RNA bands did appear, which gives the false impression of being unable to analyze the results of the bioanalyzer integrity. Low RIN number RNA samples were further processed by RNA-sequencing RNA-seq experiments from guar. RNA-seq analysis via Illumina's paired-end method was carried out to trace back and evaluate the extracted total RNA, resulting in high-quality data for subsequent stress tolerance research.

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Section: Resultsmentioning

confidence: 99%

High RNA quality extracted from the tolerant crop Cyamopsis tetragonoloba (L.) despite possession of low RNA integrity number

Gaafar

Al‐Qurainy

Alshameri

et al. 2021

Biotechnology & Biotechnological Equipment

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show abstract

“…Nowadays, RNA quality is usually assessed by quantification of RNA on ethidium bromide gels (Jakovljevic et al 2010;Tavares et al 2011;Sambrook and Russell 2001) or evaluated via measurement of absorbance where an A260/A230 ratio higher than 1.8 is considered as an indicator of extracted RNA with a low level of contamination (Tavares et al 2011;Pester et al 2012). In the study presented here, we selected the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) as the most suitable and dedicated method allowing for precise measurements of RNA.…”

Section: Resultsmentioning

confidence: 99%

“…In the study presented here, we selected the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) as the most suitable and dedicated method allowing for precise measurements of RNA. Evaluation based on electrophoresis where the sharpness of product visible in the gel is stated as good quality (Jakovljevic et al 2010) seems imprecise, as it relies on the human interpretation of a gel image (Schroeder et al 2006), but spectrometric methods are not very sensitive and do not give an answer about RNA integrity.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of different RNA extraction methods for high-quality total RNA and mRNA from Erwinia amylovora in planta

et al. 2016

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Obtaining a sufficient quantity of high-quality, intact RNA is the first crucial step in its study by RNA sequencing on next-generation sequencing platforms or quantitative PCR. Different RNA extraction methods or commercial kits vary in yield and in the quality and integrity of the RNA obtained, which may affect the results of downstream applications. Often, these factors depend on the organism under study and nature of the sample. Therefore, the selection of an appropriate RNA isolation method is critical. In this study, we present the results of an evaluation of three different commercial kits for the isolation of total RNA from Erwinia amylovora in apple tissue as well as the usefulness of different kinds of Deoxyribonuclease I for DNA removal and kits for rRNA depletion. To our knowledge, this is the first report on the method of isolation of high-quality E.amylovora mRNA for RNA-seq.

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“…The TRI reagent (Invitrogen, Carlsbad, CA, USA) is based on this technology and rapidly inhibits RNase activity, making it a complete, ready-to-use reagent for total RNA extraction (Kang et al 2011; Jakovljevic et al 2010). …”

Section: Introductionmentioning

confidence: 99%

“…The most common RNA extraction kits for frozen blood are based on the use of an acid guanidinium thiocyanate-phenol-chloroform extraction reagent. The TRI reagent (Invitrogen, Carlsbad, CA, USA) is based on this technology and rapidly inhibits RNase activity, making it a complete, ready-to-use reagent for total RNA extraction (Kang et al 2011 ; Jakovljevic et al 2010 ).…”

Section: Introductionmentioning

confidence: 99%

Comparison of three different kits for extraction of high-quality RNA from frozen blood

et al. 2014

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Extraction of high-quality RNA is a crucial step in gene expression profiling. To achieve optimal RNA extraction from frozen blood, the performance of three RNA extraction kits- TRI reagent, PAXgene blood RNA system (PAXgene) and NucleoSpin RNA blood kit (NucleoSpin)- was evaluated. Fifteen blood specimens collected in tubes containing potassium ethylenediaminetetraacetic acid (EDTA) and stored at −80°C for approximately 5 years were randomly selected. The yield and purity of RNA, RIN (RNA integrity number) values and cycle threshold (Ct) values were assessed. Mean RNA yields with TRI reagent, PAXgene and NucleoSpin were 15.6 ± 8.7 μg/ml, 3.1 ± 1.7 μg/ml and 9.0 ± 5.5 μg/ml, respectively. Mean A260/280 ratios of RNA for the three kits were 1.7 ± 0.1, 2.0 ± 0.1, and 2.0 ± 0.0, and mean RIN values recorded as 3.2 ± 0.8, 6.0 ± 1.1, and 6.4 ± 0.9, respectively. The Ct values of housekeeping genes, 18S rRNA, β-actin, RPLP0 and HPRT1, were as follows: TRI reagent (19.2 ± 1.6, 30.6 ± 1.8, 29.9 ± 1.4 and 36.3 ± 1.3), PAXgene 16.6 ± 1.4, 26.4 ± 1.3, 28.2 ± 1.8 and 33.8 ± 1.1), and NucleoSpin (16.3 ± 1.5, 27.2 ± 1.3, 27.0 ± 1.6 and 32.9 ± 1.6). RNA yield using TRI reagent was 1.7 times higher than that with NucleoSpin and 5 times higher than that with PAXgene. However, the purity and integrity of TRI-extracted RNA was lower than that extracted with PAXgene and NucleoSpin. Moreover, the Ct values of housekeeping genes after extraction with TRI reagent were approximately 1.7-3.8 times higher than those obtained with PAXgene and NucleoSpin. The PAXgene and NucleoSpin kits produced similar results in terms of RNA purity and integrity and subsequent gene amplification. However, RNA yields from NucleoSpin were 2.9-fold higher, compared to PAXgene. Based on these findings, we conclude that NucleoSpin is the most effective kit for extraction of abundant and high-quality RNA from frozen blood.

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Comparison of phenol-based and alternative RNA isolation methods for gene expression analyses

Cited by 4 publications

References 6 publications

High RNA quality extracted from the tolerant crop Cyamopsis tetragonoloba (L.) despite possession of low RNA integrity number

High RNA quality extracted from the tolerant crop Cyamopsis tetragonoloba (L.) despite possession of low RNA integrity number

Evaluation of different RNA extraction methods for high-quality total RNA and mRNA from Erwinia amylovora in planta

Comparison of three different kits for extraction of high-quality RNA from frozen blood

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