The Experts below are selected from a list of 115929 Experts worldwide ranked by ideXlab platform
Nicoletta Sacchi - One of the best experts on this subject based on the ideXlab platform.
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the single step method of RNA Isolation by acid guanidinium thiocyanate phenol chloroform extraction twenty something years on
Nature Protocols, 2006Co-Authors: Piotr Chomczynski, Nicoletta SacchiAbstract:The single-step method of RNA Isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on
Piotr Chomczynski - One of the best experts on this subject based on the ideXlab platform.
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the single step method of RNA Isolation by acid guanidinium thiocyanate phenol chloroform extraction twenty something years on
Nature Protocols, 2006Co-Authors: Piotr Chomczynski, Nicoletta SacchiAbstract:The single-step method of RNA Isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on
Katherina Sewald - One of the best experts on this subject based on the ideXlab platform.
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RNA Isolation from precision-cut lung slices (PCLS) from different species
BMC research notes, 2017Co-Authors: Monika Niehof, Tobias Hildebrandt, Olga Danov, Kirsten Arndt, Jeannette Koschmann, Franziska Dahlmann, Tanja Hansen, Katherina SewaldAbstract:Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research. To enable wider implementation in research and development, these new biologically relevant organ models are being constantly refined. A very important issue is to improve the preparation of high-quality RNA (ribonucleic acid) from PCLS for drug discovery and development of new therapies. Gene expression analysis at different levels is used as an important experimental readout. Genome-wide analysis using microarrays is mostly applied for biomarker selection in disease models or in comprehensive toxicological studies. Specific biomarker testing by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is often used in efficacy studies. Both applications require high-quality RNA as starting material for the generation of reliable data. Additionally, a small number of slices should be sufficient for satisfactory RNA Isolation to allow as many experimental conditions as possible to be covered with a given tissue sample. Unfortunately, the vast amount of agarose in PCLS impedes RNA extraction according to the standard procedures. We established an optimized protocol for RNA Isolation from PCLS from humans, rats, mice, marmosets, and rhesus macaques based on the separation of lysis and precipitation steps and a magnetic-bead cleanup procedure. The resulting RNA is of high purity and possesses a high degree of integrity. There are no contaminations affecting RTqPCR efficiency or any enzymatic step in sample preparation for microarray analysis. In summary, we isolated RNA from PCLS from different species that is well suited for RTqPCR and for microarray analysis as downstream applications.
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RNA Isolation from precision-cut lung slices (PCLS) from different species
BMC Research Notes, 2017Co-Authors: Monika Niehof, Tobias Hildebrandt, Olga Danov, Kirsten Arndt, Jeannette Koschmann, Franziska Dahlmann, Tanja Hansen, Katherina SewaldAbstract:Background Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research. To enable wider implementation in research and development, these new biologically relevant organ models are being constantly refined. A very important issue is to improve the preparation of high-quality RNA (ribonucleic acid) from PCLS for drug discovery and development of new therapies. Gene expression analysis at different levels is used as an important experimental readout. Genome-wide analysis using microarrays is mostly applied for biomarker selection in disease models or in comprehensive toxicological studies. Specific biomarker testing by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is often used in efficacy studies. Both applications require high-quality RNA as starting material for the generation of reliable data. Additionally, a small number of slices should be sufficient for satisfactory RNA Isolation to allow as many experimental conditions as possible to be covered with a given tissue sample. Unfortunately, the vast amount of agarose in PCLS impedes RNA extraction according to the standard procedures. Results We established an optimized protocol for RNA Isolation from PCLS from humans, rats, mice, marmosets, and rhesus macaques based on the separation of lysis and precipitation steps and a magnetic-bead cleanup procedure. The resulting RNA is of high purity and possesses a high degree of integrity. There are no contaminations affecting RTqPCR efficiency or any enzymatic step in sample preparation for microarray analysis. Conclusions In summary, we isolated RNA from PCLS from different species that is well suited for RTqPCR and for microarray analysis as downstream applications.
Balazs Antus - One of the best experts on this subject based on the ideXlab platform.
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Elimination of bacterial DNA during RNA Isolation from sputum: Bashing bead vortexing is preferable over prolonged DNase treatment.
PloS one, 2019Co-Authors: Csilla Paska, Imre Barta, Orsolya Drozdovszky, Balazs AntusAbstract:Sputum often contains large amounts of contaminating bacterial DNA that, if not eliminated during RNA Isolation, may interfere with gene expression studies. During RNA Isolation only repeated DNase treatment can effectively remove contaminating bacterial DNA from samples, but this compromises RNA quality. In this study we tested alteRNAtive methods to facilitate the removal of DNA and improve the quality of RNA obtained. Sputum samples obtained from patients with chronic obstructive pulmonary disease were processed with dithiothreitol and subjected to various RNA Isolation methods, yet with modified protocols. Modifications included prolonged DNase treatment or vortexing of sputum cells in the presence of beads prior to RNA Isolation. Bacterial DNA contamination was tested by PCR using universal bacterial primers, while RNA quality was assessed by real-time PCR using GAPDH primers for amplicons of different length. We found that the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column was able to completely eliminate bacterial DNA, if sputum cells were lysed in the presence of bashing beads. Notably, compared with the standard protocol, the modified procedure yielded better quality RNA as well, as indicated by improved threshold profiles of qPCR. Bead vortexing of cells was less effective when combined with other RNA Isolation methods, and the repeated DNase treatment needed to completely remove contaminating DNA from the samples reduced the quality of RNA markedly. Bead vortexing in combination with certain RNA extraction methods greatly facilitates the Isolation of sputum RNA that is free of contaminating bacterial DNA, and is suitable for downstream applications.
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Pitfalls of RNA Isolation from sputum in COPD
European Respiratory Journal, 2014Co-Authors: Csilla Paska, Imre Barta, Krisztina Kelemen, Balazs AntusAbstract:Aims: Sputum could be a valuable source for molecular analysis of respiratory diseases; however, various methodological problems have to be overcome. We attempted to maximize the quality and the quantity of RNA by optimizing sputum processing, RNA Isolation and transcription methods. Methods: Sputum samples (n=20) were obtained from COPD patients at our Institute, and processed using dithiothreitol (DTT), n-acetyl cysteine or RNAlater solutions. Three different RNA Isolation methods were tested. DNA contamination was tested by reverse transcription negative controls with GAPDH and universal bacterial primers. RNA quality was assessed by real-time PCR yielding GAPDH amplicons of different length. Data are presented as mean±SD. Results : Sputum cell counts varied between 0.95-7´10 6 cells/g sputum. Isolated RNA content was 332.9±322.2 ng/g sputum. All three processing methods yielded similar amount of RNA, with a slight advantage for DTT (p=NS). Cell integrity was only preserved in DTT. DNase treatment diminished the yield by 30-50%. Surprisingly, in most samples several rounds of DNase treatment was needed to clean the samples from the contaminating DNA. The choice of RNA Isolation kit, the speed and the temperature of centrifugation significantly influenced the amount of RNA. The choice of reverse transcriptase had a lesser effect on the PCR products. Conclusion: The quality and the quantity of sputum RNA depends on several factors during the Isolation. However, the biggest challenge is the elimination of bacterial DNA, which is of high importance, since contaminating bacterial background might mask the human RNA in gene expression studies.
Yijun Zhou - One of the best experts on this subject based on the ideXlab platform.
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Development of a simplified RT-PCR without RNA Isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén).
Virology journal, 2017Co-Authors: Haoqiu Liu, Pingping Yuan, Xiaoxia Zhang, Qingqing Chen, Xuanli Jiang, Yijun ZhouAbstract:The small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA Isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA Isolation when processing a large number of samples. We established a simplified RT-PCR method without RNA Isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 103 fold. Compared to the traditional RT-PCR with RNA Isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA Isolation. A simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA Isolation step. It offers a convenient alteRNAtive to the traditional RT-PCR method.
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Development of a simplified RT-PCR without RNA Isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén)
BMC, 2017Co-Authors: Haoqiu Liu, Pingping Yuan, Xiaoxia Zhang, Qingqing Chen, Xuanli Jiang, Yijun ZhouAbstract:Abstract Background The small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA Isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA Isolation when processing a large number of samples. Results We established a simplified RT-PCR method without RNA Isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 103 fold. Compared to the traditional RT-PCR with RNA Isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA Isolation. Conclusions A simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA Isolation step. It offers a convenient alteRNAtive to the traditional RT-PCR method
