Reverse Transcriptase Pcr
Reverse Transcriptase Pcr - Related Videos
03:35
Assessing Viral Replication Capacity Using a Radiolabeled Reverse Transcriptase Assay
0 Views •
2026
Source: Claiborne, D. T. et al. A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses. J. Vis. Exp. (2014)This video demonstrates the assessment of viral replication capacity using a radiolabeled reverse transcriptase assay. Culture supernatants containing progeny virions are added to a reverse transcription mixture containing a radiolabeled nucleotide. The incorporated signal reflects viral output and replication...
03:26
Reverse Transcriptase Activity as a Marker for Virus-Like Particle Production
0 Views •
2026
Source: Scarborough, R. J., et al. Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy. J. Vis. Exp. (2016).This video demonstrates a reverse transcriptase (RT) assay to assess the effect of a test RNA on virus-like particle (VLP) production. Cells are co-transfected with plasmids, and RT activity is measured as a quantitative indicator of VLP output. A decrease in RT activity reflects suppression of viral gene expression by the test RNA.
04:08
Real-Time Quantitative Reverse Transcription PCR for Diagnosing Viral Infections
0 Views •
2025
This video demonstrates a quantitative method for detecting viral infection using real-time reverse transcription PCR. The reverse transcription step converts the RNA to cDNA, which is then amplified via PCR cycles. The presence of viral RNA in the sample is confirmed by analyzing the amplification and dissociation curves of DNA obtained from the PCR.
04:52
Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
0 Views •
Cited by 11 •
2011
We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.
05:46
Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
0 Views •
Cited by 15 •
2014
Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.
FAQs
cDNA provides a DNA form of the original RNA sequence for the amplification stage. Because reverse transcriptase uses the RNA template before polymerase chain reaction begins, the method can retain information from RNA molecules that reflects gene expression or an RNA-based pathogen. This ordering links the initial RNA target to the later amplified product, making the conversion step essential for interpreting the assay.
Each cycle first separates the DNA strands through denaturation, then allows primers to anneal to their selected sequences, and ends with DNA extension. Repeating this sequence increases the amount of the chosen target, rather than broadly copying all nucleic acids in the sample. Primer selection therefore determines which transcript or pathogen-derived sequence the assay examines.
An RNA target requires the conversion step because the amplification stage operates on cDNA. In this sense, reverse transcriptase PCR differs from a DNA-focused PCR workflow, which would not need reverse transcriptase to create cDNA from RNA. This distinction matters when the biological question concerns gene expression or an RNA-based pathogen rather than a DNA sequence.
A basic workflow begins with an RNA template and reverse transcriptase synthesis of cDNA. The resulting cDNA then enters PCR, where denaturation, primer annealing, and DNA extension occur repeatedly. The selected target is amplified during these cycles, producing material for analysis of the RNA-derived sequence. This two-stage order keeps RNA conversion conceptually separate from amplification.
In immunology and infection studies, RT-PCR can examine cytokine transcripts and other immune-gene transcripts. Comparing these transcript signals across samples or conditions can help investigators assess host responses associated with infection. This use connects molecular RNA measurements with questions about immune activation and host-pathogen interactions, while keeping the assay focused on selected genes.
For RNA-based pathogens, RT-PCR can identify viral genomes and support infection diagnosis and disease surveillance. Repeated or comparative measurements can also monitor changes in pathogen burden or in host response transcripts. These outputs provide complementary information: one concerns detection of viral genetic material, while the other concerns transcriptional changes associated with the infection.