Yield and Integrity of RNA from Brain Samples are Largely Unaffected by Pre-analytical Procedures
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Brain tissue
Human brain
The existence of metabolites that interfere with isolation procedures and downstream applications makes plant RNA extraction difficult. The current research used a standardized RNA extraction protocol from maize (Zea mays L.). We developed a protocol for extracting pure RNA from plant tissues using both extraction buffer and the TRIzol reagent, and show that this RNA extraction method works not only at Low temperatures but also at room temperatures, making it the easiest and most efficient method for extracting pure and undegraded RNA directly from tropical plants in the field. RNA isolation methods based on our modified protocol yielded good results in maize leaf, seed, flowers and other grass species. The isolated RNA was found to be suitable for both PCR and RT-PCR amplification and transcriptome analysis. The method is repeatable and can be used to isolate high-quality RNA and conduct gene expression studies. RNA extraction paves the way for deciphering the complex regulatory network involved in multiple stress responses by studying gene-environment interactions at the transcriptome level.
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Human brain
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Human brain
Brain tissue
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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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Agarose gel electrophoresis
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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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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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Agarose gel electrophoresis
Agarose
28S ribosomal RNA
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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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Agarose gel electrophoresis
Cloning (programming)
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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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Isolation
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