RT-qPCR
Definition
Reverse-transcription quantitative PCR: a two-step assay in which RNA is first converted to complementary DNA (cDNA) by reverse transcriptase, then amplified and quantified by real-time PCR. The cycle threshold (Ct) value is inversely proportional to the initial RNA abundance.
- Full name
- Reverse-transcription quantitative PCR
- Step 1
- RNA converted to cDNA by reverse transcriptase
- Step 2
- Real-time PCR amplification and quantification
- Key output
- Cycle threshold (Ct) value
Common questions
How is Ct value used to estimate stain age?+
Ct value is inversely related to how much starting RNA was present, and RNA degrades over time in a bloodstain or other fluid, so tracking Ct or ratios of stable to unstable transcripts can give a relative estimate of how long a stain has aged, though it is not a precise clock.
Why use RT-qPCR rather than end-point RT-PCR for this application?+
Real-time quantification during amplification gives a numeric Ct value for comparison between samples, which end-point PCR cannot provide since it only shows whether a product formed, not how much starting material was there.
Related terms
- Degradation Index
- A ratio or combination of RNA abundance measurements designed to capture the state of degradation of a sample. Often calculated as the...
- Inter-Individual Variability
- Differences in RNA degradation rate between different donors' blood, arising from variation in RNase activity, blood cell composition, and haematological status. One...
- microRNA (miRNA)
- Small non-coding RNA molecules of approximately 18-24 nucleotides that regulate gene expression post-transcriptionally. Abundant in blood cells, relatively resistant to degradation compared...
- mRNA (Messenger RNA)
- Transcripts encoding proteins, synthesised in the nucleus from DNA templates and translated by ribosomes. mRNA molecules vary greatly in length (hundreds to...
- RNase
- Ribonuclease enzymes that cleave RNA phosphodiester bonds. Present in blood plasma (RNase A family) and released from cells on death. The activity...