How to measure RNA level?
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21 Citations | In addition, this assay can be used to measure the changes in RNA concentration in real-time and to quantify short RNAs (<30 nucleotides). |
17 Citations | The fluorescence stopped-flow assay, therefore, provides a high-throughput way to measure the kinetic parameters of RNA synthesis. |
56 Citations | Quantitative in situ RNA measurement at the single-cell level may be broadly applicable in companion diagnostic applications. |
77 Citations | Changes in RNA secondary structure emerge as a possible explanation necessitating the development of methods to measure the impact of single-nucleotide variation on RNA structure. |
| In summary, our workflow is a versatile approach to RNA modification level estimation, which is open to any read-count-based experimental approach. | |
7 Citations | This sensitivity permits RNA base analysis at the cellular level. |
| Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell. | |
6 Citations | Our assay provides a fast and accurate quantification method to measure changes in Ψ levels of different RNA pools without sample derivatization. |
50 Citations | A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. |
56 Citations | We furthermore demonstrate that above a certain threshold of test mRNA level changes and given high quality RNA processing, normalization to total RNA alone provides for equally reliable quantitative mRNA level results. |
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How to measure the RNA modification?4 answersMeasuring RNA modifications is a multifaceted process that involves various techniques and technologies, each contributing uniquely to the field of epitranscriptomics. One foundational approach is the use of mass spectrometry, which allows for the preparation of samples from biological specimens to measure both intracellular and extracellular RNA modifications. This technique is particularly powerful for identifying epitranscriptomic biomarker signatures in cancer diagnosis, as it can reliably quantify RNA marks in solid biopsies and biological fluids.
Direct RNA sequencing from Oxford Nanopore Technologies (ONT) represents another comprehensive method for detecting RNA modifications. It faces informatics challenges due to the complexity of the data, but software like nanoDoc2, which includes a novel signal segmentation algorithm and a machine learning component, has been developed to accurately detect modifications from nanopore direct RNA sequencing data. Similarly, xPore offers a computational method and statistical framework for analyzing differential RNA modifications from nanopore data, while Nanocompore provides an analytical framework to evaluate the presence of modifications in direct-RNA sequencing data by comparing RNA samples against non-modified controls.
Technological innovations have also contributed to the high-resolution detection of RNA modifications, such as the engineered MspA nanopore. Additionally, direct chemical modification and measurement of RNA sequences through capillary electrophoresis and mass spectrometry offer a method to overcome the limitations of indirect RNA sequencing technologies.
NMR spectroscopy, sequencing, and mass spectrometry of the epitranscriptome have been instrumental in revealing the function of modified residues, while the mapping of RNA modifications has been significantly advanced by deep sequencing-based methods and protocols. Each of these methods and technologies plays a crucial role in the precise mapping and quantification of RNA modifications, contributing to our understanding of their roles in cellular processes and disease.
Why is RNA extracted and quantified?4 answersRNA is extracted and quantified for several reasons. Accurate quantification of RNA is important for downstream RNA analysis techniques. It helps in determining the concentration of RNA in a sample, which is necessary for performing optimal RNA analysis. Different methods, such as UV spectrophotometric and fluorescent dye-based methods, are used for quantifying RNA depending on the purity and concentration of the sample. RNA extraction is also necessary for cell characterization and studying gene expression. In the context of infection diagnosis, measuring RNA expression profiles can provide a specific systemic inflammatory response to infection, which can be used as a molecular diagnostic approach. Additionally, accurate RNA quantification is important for quality control in RNA synthesis and extraction processes. Overall, RNA extraction and quantification are essential steps in various research and diagnostic applications.
Why is RNA quantified after extraction?5 answersRNA quantification is important after extraction because it allows for accurate measurement of the concentration of RNA in a sample, which is necessary for downstream RNA analysis techniques. There are two main methods for quantifying RNA: UV spectrophotometric methods and fluorescent dye-based methods. UV spectrophotometric methods are simple and accurate when the RNA sample is pure, but fluorescent dye-based methods are better for samples with impurities or low RNA concentrations. Proper quantification of RNA is crucial for ensuring the reliability and reproducibility of downstream gene expression analysis. It helps to determine the amount of RNA available for analysis and can aid in minimizing sample degradation. Accurate quantification also allows for the optimization of downstream protocols and the standardization of experimental procedures. Overall, RNA quantification is essential for obtaining reliable and meaningful results in molecular biology research.
How does biodrop spectrophotometer calcularte RNA concentration?4 answersThe BioDrop spectrophotometer calculates RNA concentration by analyzing UV-VIS spectrophotometer data and fitting it with spectra representative of base pairs such as adenine-thymine (AT) or guanine-cytosine (GC) or adenine-uracil. From this fitting process, the BioDrop spectrophotometer derives a quantification of the amount of DNA and/or RNA present in the sample.
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