A quick and cost-effective method for DNA-free total RNA isolation using magnetic silica beads
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ABSTRACT Current RNA purification methods widely use silica-based columns that allow quick isolation of high-quality and good quantities of RNA. However, the major limitations include high cost, the requirement of different kits for small RNA isolation, genomic DNA contamination, and not being flexible. Here, we used the in-house RNA isolation reagent (RIR) for cell lysis, followed by RNA precipitation using isopropanol. RNA isolated using the in-house RIR resulted in a similar quantity and quality compared to the commercial TRIzol. Furthermore, the commercial RNA isolation kits with silica-based columns recommend genomic DNA digestion during or after RNA purification, adding time and cost to RNA purification. Here, we developed an optimized in-house protocol for isolating high-quality RNA free of genomic DNA contamination using magnetic silica beads without needing DNase digestion. Additionally, our method purifies total RNA along with the small RNA fraction, including miRNAs, which usually require a separate kit for extraction. Additionally, the RNA prepared with our method was equally suitable for mRNA and miRNA expression analysis using RT-qPCR. Together, the in-house method of RNA isolation using the magnetic silica beads has exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.Keywords:
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Objective To explore an optimal method for the extraction of high quality RNA in microarray analysis by comparing and improving Trizol and two common RNA extraction kits methods for RNA extraction from mouse brain. Methods Total RNA of mouse brain was isolated with the Trizol,Ambion and Tiangen tissue RNA extraction kits,and RNA extraction kits methods were optimized.The concentration and purity of RNA and cRNA were determined by ultraviolet spectrophotometer,and the integrity was determined by agarose gel electrophoresis. Results The optimized procedure for RNA extraction displayed higher yield of total RNA. The purity of RNA with Ambion kit method was better than Trizol and Tiangen kit method. Conclusion The concentration and purity of RNA by optimized procedure of Ambion kit are better than those by other methods,and are suitable for microarray analysis.
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Isolation of high-quality RNA from seeds is very critical for seed specific gene analysis. An investigation and comparison on the use of CTAB, SDS and TRIzol extraction procedures to extract high-quality RNA from grains of medical njavara rice were carried out in the present study. These protocols either failed to yield RNA or resulted in reduced yield with poor quality of RNA from Njavara rice grains. The starch and secondary metabolites present in njavara rice seeds hindered the isolation and the resuspension of precipitated RNA or contaminated the RNA pellets by co-precipitation. Hence, we modified the TRIzol RNA extraction protocol by addition of 0.5% N-lauryl sarcosine, 2% β-Mercaptoethanol and 1% PVP. Highly pure (A260/A280 ratio ranged from 1.9 to 2.0) and intact RNA with a higher yield (up to 500µg/ml of RNA) could be obtained using modified TRIzol RNA extraction protocol. RNA obtained was testified and efficiently utilized for the cDNA preparation and amplification of Ubiquitin gene.
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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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Isolation of high-quality RNA is a prerequisite to study the development of wheat seeds at the gene expression level.Rapid isolation of high-quality total RNA from wheat seeds is difficult with the extraction methods reported previously.In the present study,cold phenolic method and Trizol single-step method were combined for RNA extraction,and high-quality total RNA was obtained in about five hours.The quality of total RNA was analyzed with agarose gel electrophoresis and UV spectrophotometer.Clear bands of 28S rRNA and 18S rRNA were shown in the RNA electrophoresis,and the value of OD260/OD280 was 1.90 to 2.00.Using the total RNA isolated by this method for reverse transcription,high quality of cDNA could be obtained,and clear bands were detected in subsequent cDNA-AFLP analysis.These results demonstrated that the purity and integrality of total RNA isolated with the new method were significantly satisfactory for the demands of molecular biological experiments.
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The first step of Insect-resistant cotton leaves BT gene cloning technology is extracting the total RNA from the insect-resistant in BT gene-specific expression of the young leaves;Gene expression can be generated in different stages of RNA to control,purification of RNA integrity is a study of gene expression and cDNA cloning of new genes from the foundation.In this study,comparative analysis of the CTAB-licl law,modified one-step guanidine isothiocya- nate,One-step Trizol method and the RNA extraction kit.Bands by agarose gel electrophoresis results of testing and UV detection,it is very difficult prior to the discovery of two methods to extract the desired RNA,RNA extraction and then the integrity of the two better,but the RNA extraction kit is more suitable for reverse transcreption cDNA.
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Quantitative PCR (qPCR) analysis of gene expression in single bovine oocytes or blastomeres from early preimplantation embryos needs to meet optimized requirements for the isolation of the RNA, the reverse transcription reaction, and even the qPCR. Normally, large amounts of tissues are required for extracting RNA, but the RNA recovery per embryo is too low for a reliable detection of low expressed genes. Therefore an optimized isolation method is essential for obtaining sufficient RNA recoveries of (a) a group of embryos/oocytes (n = 10), (b) a single oocyte/embryo, or (c) single blastomeres without inhibition of the subsequent steps. For each experiment studied, the RNA was reverse-transcribed with ExtremeScript-OLS� (Omni Life Science, Inc., Raynham, MA, USA) following the manufacturer's instructions. The transcript levels were analyzed by the detection of the genes STAT3 and LEPR. First, we compared different isolation protocols from the literature for the isolation of oocyte RNA[TriZol� (Invitrogen, Carlsbad, CA, USA) and RNAPure (Peqlab Biotechnologie, Erlangen, Germany) for isolation of total RNA; magnetic beads and Absolutely RNA Nanoprep (Stratogene, La Jolla, CA, USA) for mRNA; n = 12 per protocol] by measurement of the total RNA concentration (TriZol and RNAPure) and qPCR analysis (all isolation techniques). The results showed that RNAPure provided the highest transcript numbers (100% RNAPure v. 78% TriZol, 25% Nanoprep, and 5% magnetic beads, respectively; P ≤ 0.001) and also the highest total RNA concentration (2.1 ng µL–1 v. TriZol 1.5 ng µL–1 total RNA per oocyte; P ≤ 0.05). In the second experiment, we analyzed the influence of a coprecipitant [glycogen, linear acrylamide, SeeDNA (Amersham Biosciences, Freiburg, Germany); n = 12] on the RNA recovery and the inhibition of the subsequent reverse transcription and PCR processes. In the third experiment, the collection/storage of single oocytes was compared [RNAlater� (Applied Biosystems, Darmstadt, Germany) or liquid nitrogen; n = 20]. The use of the coprecipitant linear acrylamide (100% v. 80% for glycogen and 58% for SeeDNA; P ≤ 0.05) and the storage in liquid nitrogen (100% v. 84% for RNAlater; P ≤ 0.001) showed the highest RNA recoveries without inhibition. Furthermore, we analyzed (with the now optimized protocol) single blastomeres derived from 8- (n = 6) and 16-cell (n = 6) IVF embryos by mechanical treatment. The aim of this experiment was to analyze the expression divergences in blastomeres from normal cultured embryos without synchronization and, further, how many single blastomeres per embryo are required to obtain a comparable expression level of all blastomeres. The results showed that nearly 50% of an embryo was required (at least 4 blastomeres from an 8-cell and 7 blastomeres from a 16-cell embryo). These findings are mainly caused by different cell cycle phases of the analyzed blastomeres. Further studies with synchronized blastomeres are in progress. In conclusion, the present study demonstrates an effective RNA isolation method for a reliable qPCR analysis of single blastomeres. This work was supported by grant of the Deutsche Forschungsgemeinschaft (DFG) (FOR 478/1).
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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.
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TRIzol Reagent,RNAplant and TRIpure Reagent were used for RNA extraction from 3 tomato tissues, including leaf,stem and root.Total RNA were evaluated upon quality and yields.RNAplant was found to be the best one among the 3 reagents evaluated.With this reagents,higher amount and quality of RNA could be successfully extracted from all 3 kinds of tissues tested.The RNA obtained from all 3 reagents were successfully used to amplify cDNA fragments by RT-PCR .
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