Comparison of RNA extraction methods for effective isolation of bacterial RNA from neisserial cultures
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Abstract:
RNA is an important macromolecule isolated in many areas of research to look at biological reactions,
gene expression, cellular signals, processing, and more. However, in order to look into specific areas of
interest, purification is required before any type of downstream transcriptomics work, such as sequencing,
microarrays, and quantitative RT-PCR, to ensure reliability and success. In this research, three different
RNA extraction methods were compared using both widely available reagents as well as commercial kits.
Each method was compared to establish how efficient and effective it was in extracting pure RNA using
the Gram-negative bacterial species, Neisseria gonorrhoeae strain NCCP11945. RNA was extracted from
cultures using the Ambion® RiboPure™ Kit (Life Technologies™), the RNeasy Kit (QIAGEN®) and a previously reported combined method using TRIzol® Reagent (Life Technologies™) followed by the RNeasy
Kit. Evaluation of RNA quantity and purity was carried out using the Nanovue® spectrophotometer and
quality and integrity was established using the Agilent 2100 Bioanalyzer and agarose gels. There was little
success with the Ambion® RiboPure™ Kit for extraction of N. gonorrhoeae RNA. Although the TRIzol/
RNeasy-based extraction was successful at producing high yields it used hazardous chemicals and required
further purification methods. It was concluded that the RNeasy kit used on its own was proven to be the
best method of extraction in terms of RNA yield, quality, reproducibility, and convenience.Keywords:
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This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.
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The isolation of high quality RNA is a crucial technique in plant molecular biology. The quality of RNA determines the reliability of downstream process like real time PCR. In this paper, we reported a high quality RNA extraction protocol for a variety of plant species. Our protocol is time effective than traditional RNA extraction methods. The method takes only an hour to complete the procedure. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The extracted RNA was further used for cDNA synthesis, expression analysis and copy number determination through Real Time PCR. The results indicate that RNA was of good quality and fit for real time PCR. This high throughput plant RNA extraction protocol can be used to isolate high quality RNA from diverse plants for real time PCR and other downstream applications.
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This paper elaborates the extraction of RNA and some points needing attention, and compares three RNA extraction kits used commonly in lab.
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ABSTRACT The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world.
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RNA extraction is a prerequisite technique for gene expression studies, analyzing the etiology and disease progression, treatment effects, as well as designing the diagnostic methods. Although many RNA extraction kits have been commercialized, but these kits are expensive and are not accessible in some countries. Many studies have shown that TRIzol is an applicable material for the RNA extraction from various biological samples. In this study, evaluation of the initial TRIzol volume and -20˚C temperature on the purity and solubility of RNA, which followed by measurement of IL-1B expression shows that TRIzol based RNA extraction is a “reliable” method for gene expression studies.
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RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit–blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.
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A11 Roche Molecular Systems has partnered with external pharmaceutical companies to develop clinical diagnostic assays that may provide patients with improved therapies. Companion diagnostics utilizing valid biomarkers coupled with certain drugs may be used for the purpose of patient stratification. In order to facilitate the development of companion diagnostic gene expression tests, a total RNA extraction method has been optimized for formalin-fixed paraffin embedded (FFPE) tissue and a qRT-PCR gene expression assay has been evaluated on the COBAS® TaqMan 48 Analyzer.Archived formalin-fixed paraffin-embedded tissue samples are an invaluable resource for gene-expression profiling in cancer patients. FFPE tissue samples are often readily available and can be associated with clinical outcome. However, extraction of quality RNA from FFPE tissue has proven to be problematic due to cross-linking of proteins and nucleotides during the formalin fixation process. We have evaluated several FFPE tissue RNA extraction kits including the High Pure RNA Paraffin Kit (Roche Applied Science) and the RNeasy® FFPE Kit (Qiagen). Based on the initial evaluation, the RNeasy® FFPE Kit was selected for further studies due to shorter processing time of 90 minutes compared to 24 hours for the High Pure RNA Paraffin Kit. Both kits had comparable performance based on amount of RNA extracted and the ability to amplify this RNA.Analytical performance of a multiplex qRT-PCR gene expression assay using COBAS® TaqMan 48 Analyzer for real-time single-tube reverse transcription, amplification and detection was evaluated. Linearity studies demonstrated R 2 values were >0.97 for the all four genes of the multiplex reaction when testing 2-fold serial dilutions (20 -0.156 ng) of Universal Human Reference RNA (Stratagene). Furthermore, assay linearity and precision using micro-dissected clinical samples extracted with the RNeasy® FFPE Kit will be presented. The current results indicate that we have a fast, non-organic, IVD compatible RNA extraction method for FFPE tissue that can provide quality RNA for multiplex real time qRT-PCR assays on the COBAS® TaqMan 48 Analyzer.
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RNA extraction is a crucial step for monitoring gene expression. Poor RNA quality (including degradation and remaining impurities) can result in misleading results. Isolation of RNA from animal tissues with high lipid content can be challenging. Especially, it is not trivial to isolate high quality RNA with a reasonable yield from adipose tissue. The aim of this study was to provide an optimized protocol for isolating total RNA from adipose tissue. This was achieved by combining the advantages of the two routinely used methods, TRI Reagent® and miRNeasy. The miRNeasy method results in cleaner samples but more prone to degradation while the TRI Reagent® method results in samples contaminated with salts and solvents but more intact. The new protocol combines the best of both methods resulting in RNA of high quality and suitable for downstream experiments like RT-qPCR, microarrays and high-throughput sequencing. The current protocol for total RNA isolation from adipose tissue yields sufficient amount of high quality total RNA free of contaminants.
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