Scenario:
we purified some samples of RNA and decided to verify the quality and quantification using two different equipments: Qubit 4 and Bioanalyzer 2100 and we got results very different for the same sample, on each equipment. That happened with all the samples that were analysed.
If this is ok, which of these two equipments is the best to do RNA quantification?
Othman Mueen Mohammed
Methodology Differences:Qubit 4: This instrument uses a fluorometric method to quantify RNA. It relies on specific dyes that bind to RNA, providing a direct measurement of RNA concentration. The Qubit 4 is known for its high sensitivity and specificity, as the dyes bind selectively to RNA, making it more accurate in distinguishing RNA from other nucleic acids like DNA 810. Bioanalyzer 2100: This system uses microfluidics-based capillary electrophoresis to separate and quantify RNA. It provides both quantitative and qualitative information about the RNA sample, including RNA integrity number (RIN) and fragment size distribution. The Bioanalyzer 2100 is less sensitive to contaminants and provides a more comprehensive analysis of RNA quality 67. Sample Volume and Handling:The Bioanalyzer 2100 typically requires a smaller sample volume (1 µL) compared to the Qubit 4, which can use a wider range of sample volumes. This difference in sample volume can lead to variations in the quantification results, especially if the sample concentration is very high or very low 3. Sensitivity and Specificity:The Qubit 4 is highly sensitive and specific to RNA, which can lead to more accurate quantification in samples with low RNA concentration. However, it may not provide information on RNA integrity or fragment size distribution 810. The Bioanalyzer 2100, while less sensitive, provides a more detailed analysis of RNA quality, including the presence of degraded RNA and the overall integrity of the sample. This can result in different quantification results, especially if the sample contains degraded RNA or other contaminants 67. Quality Metrics:The Bioanalyzer 2100 provides quality metrics such as the RNA Integrity Number (RIN), which can affect the quantification results. The presence of 5S rRNA and tRNA, which can form tertiary structures and bind to the RNA dye, can lead to differences in the quality scores between the Qubit 4 and the Bioanalyzer 2100
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