Quantification of Cy3/Cy5 labeled RNA and ssDNA
Applications | 2020 | Unchained LabsInstrumentation
Accurate quantification of Cy3 and Cy5 labeled RNA and single-stranded DNA is essential for reliable downstream applications including probe design, labeling efficiency assessment, and quality control in molecular biology workflows.
This application note outlines procedures for utilizing the RNA Cy3/Cy5 and ssDNA Cy3/Cy5 Unmix applications on Lunatic systems. It describes how to isolate the target molecule spectrum from co-absorbing contaminants and calculate concentration and degree of labeling.
The Unmix approach employs UV/Vis spectral deconvolution to separate contributions from the target nucleic acid, Cy3 and Cy5 labels, common impurities, and background scattering. Pure water is used as a blank to correct baseline absorption.
The Unmix application identifies the following spectral components:
The quality of fit is expressed as RRSE; values above 2.5 percent trigger a warning and default to total nucleic acid quantification. Samples with low A260 absorbance are similarly flagged.
The system generates comprehensive reports in HTML, XML, TXT, CSV, XLSX, and PDF formats.
The spectral unmixing strategy provides rapid, label-specific quantification without extensive sample preparation. It streamlines labeling efficiency assessment and enhances data quality for research, QC, and diagnostic laboratories.
Advancements may include application to additional fluorescent labels, integration with laboratory information management systems, automated real-time monitoring, and incorporation of advanced algorithms for improved spectral deconvolution.
The Lunatic Unmix applications offer a robust solution for precise measurement of Cy3 and Cy5 labeled nucleic acids, combining ease of use, detailed reporting, and reliable quality metrics for diverse analytical workflows.
No external references were provided in the original document.
UV–VIS spectrophotometry, Particle characterization
IndustriesProteomics
ManufacturerUnchained Labs
Summary
Importance of the Topic
Accurate quantification of Cy3 and Cy5 labeled RNA and single-stranded DNA is essential for reliable downstream applications including probe design, labeling efficiency assessment, and quality control in molecular biology workflows.
Objectives and Overview
This application note outlines procedures for utilizing the RNA Cy3/Cy5 and ssDNA Cy3/Cy5 Unmix applications on Lunatic systems. It describes how to isolate the target molecule spectrum from co-absorbing contaminants and calculate concentration and degree of labeling.
Methodology and Instrumentation
The Unmix approach employs UV/Vis spectral deconvolution to separate contributions from the target nucleic acid, Cy3 and Cy5 labels, common impurities, and background scattering. Pure water is used as a blank to correct baseline absorption.
Used Instrumentation
- Lunatic UV/Vis spectrophotometer with Unmix applications
- Little Lunatic microvolume system
Main Results and Discussion
The Unmix application identifies the following spectral components:
- Target RNA or ssDNA concentration reported in ng/µL and pmol/µL
- Cy3 and Cy5 dye amounts
- Impurities such as proteins, guanidine thiocyanate, azide, EDTA, citrate, and phenol (RNA only)
- Background contribution from turbidity and hemoglobin
The quality of fit is expressed as RRSE; values above 2.5 percent trigger a warning and default to total nucleic acid quantification. Samples with low A260 absorbance are similarly flagged.
The system generates comprehensive reports in HTML, XML, TXT, CSV, XLSX, and PDF formats.
Benefits and Practical Applications
The spectral unmixing strategy provides rapid, label-specific quantification without extensive sample preparation. It streamlines labeling efficiency assessment and enhances data quality for research, QC, and diagnostic laboratories.
Future Trends and Applications
Advancements may include application to additional fluorescent labels, integration with laboratory information management systems, automated real-time monitoring, and incorporation of advanced algorithms for improved spectral deconvolution.
Conclusion
The Lunatic Unmix applications offer a robust solution for precise measurement of Cy3 and Cy5 labeled nucleic acids, combining ease of use, detailed reporting, and reliable quality metrics for diverse analytical workflows.
Reference
No external references were provided in the original document.
Content was automatically generated from an orignal PDF document using AI and may contain inaccuracies.
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