Viral Rna Quantification
Viral Rna Quantification - Related Videos
02:31
Sample Preparation for Luciferase-Based Viral Quantification
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2026
Source: Garcia, V., et al. High-throughput Titration of Luciferase-expressing Recombinant Viruses. J. Vis. Exp. (91), e51890, doi:10.3791/51890 (2014).This video demonstrates the preparation of cell culture samples to assess viral load using a luciferase-based assay. It outlines the process of infecting epithelial cells with a genetically modified virus, transferring viral supernatants and standards into assay wells, and promoting viral entry through centrifugation and incubation. The...
10:16
Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)
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Cited by 35 •
2012
A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.
02:59
Extraction of Viral RNA from Surface-Bound Virions Using a Silica Spin Column
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2026
Source: Klaus, J. P. et al. Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment. J. Vis. Exp. (2016)This video demonstrates the extraction of viral RNA from surface-bound virions on epithelial cells using silica spin columns. The method includes chemical lysis, RNA stabilization, and membrane-based purification for downstream quantification of virus-cell attachment.
09:35
Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis
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Cited by 15 •
2013
This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.
04:23
Determining Viral RNA Distribution in Host Cells by Fluorescence In Situ Hybridization
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2026
Source: Vallery, T. K. & Steitz, J. A. Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells. J. Vis. Exp. (2019)This video demonstrates fluorescence in situ hybridization to visualize viral RNA in virus-infected cells. It outlines the steps involved in probe hybridization, signal amplification, and confocal imaging.
FAQs
Reverse transcription provides the bridge between the viral RNA template and quantitative PCR. During this step, RNA is converted into complementary DNA, which can then be amplified while fluorescence is monitored across cycles. Keeping these stages conceptually distinct helps researchers understand whether variation arises during RNA preparation, conversion to complementary DNA, or amplification.
The fluorescence signal changes as complementary DNA amplification proceeds, creating a cycle-dependent measurement of the target. Researchers compare this signal with results from standards to translate the amplification pattern into an estimated RNA concentration or copy number. This comparison makes the assay quantitative rather than simply indicating whether viral material is detectable.
Standards provide the reference needed to relate a measured amplification signal to an amount of viral RNA. By comparing the sample result with these known reference points, researchers can estimate concentration or copy number. Without that comparison, fluorescence still indicates amplification, but it does not by itself provide the same quantitative interpretation.
Sample preparation affects whether viral RNA is represented accurately in the material entering the assay, while controls help identify contamination or technical variation. These safeguards are especially important when comparing samples, because an apparent difference in viral signal may reflect handling or assay performance rather than a genuine change in viral burden.
A typical workflow begins with careful sample preparation, followed by reverse transcription to generate complementary DNA. Quantitative PCR then amplifies that material while fluorescence is recorded across cycles. Researchers compare the resulting signal with standards and examine controls before reporting an RNA concentration or copy number and assessing whether technical variation could affect the result.
The measurement supports diagnosis, infection monitoring, and experimental studies of viral replication dynamics. In immunology research, investigators can relate changes in viral RNA to host immune responses. During treatment studies or experimental infection, repeated measurements can help assess how the measured viral burden changes over time and whether those changes accompany altered biological conditions.