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1. Absolute quantitation:
Quantitation of unknown samples by interpolating their quantity from a standard curve.
2. Amplicon:
Piece of DNA formed as the product of a round of PCR (as well as ligase chain reaction (LCR) or by natural gene duplication).
3. Amplification plot:
An amplification plot is the plot of fluorescence signal versus cycle number. In the initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline for the amplification plot. An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. A fixed fluorescence threshold can be set above the baseline.
4. Baseline:
A linear function subtracted from the data to eliminate background signal.
5. Dynamic Range:
The linear range of fluorescent signal (from the lowest to the highest in the experiment) that can be detected without saturating the system. A wide dynamic range in a real-time system confers the ability to detect samples with high and low copy number in the same run. The example below showing a 10 fold dynamic range.
6. Fluorophore:
(reporter dye): The fluorescent dye used to monitor PCR product accumulation in a real-time PCR experiment. This can be attached to a probe or free in solution (e.g. SYBR® Green I).
7. Multiplex-PCR:
The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be illicited from a single test run that otherwise would require several times the reagents and time to perform.
8. Melting profile:
Control for primer dimer formation, the melting curve is created after completion of PCR by gradually increasing the temperature and measuring the fluorescence as function of the temperature.
9. NAC:
(No amplification control) will show if probes are degraded.
10. No RTC:
(No reverse transcriptase control) will show template contamination.
11. NTC:
(No template control) will show primer dimers or template contamination.
12. One-Step PCR:
(See RT-PCR): In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipeting steps and time, and is easy in handling, making it ideal for high throughput screening.
13. Qualitative Detection:
Allows you to determine the presence or absence of template of interest based on either Ct values or endpoint fluorescence.
14. Quantitative PCR (qPCR):
(see real-time PCR): PCR reaction is measured and monitored in ‘real-time’ in a closed-tube system, by measuring fluorescence intensity during each amplification cycle, to ‘quantitatively’ determine mRNA signal levels and/or DNA genes.
15. Quencher:
A compound used in real-time PCR experiments that absorbs the energy of the reporter dye in its excited state. The quencher can emit its own fluorescent signal (e.g. TAMRA) or emit no fluorescent signal (e.g., DABCYL, BHQ).
16. Real-time PCR:
(See RT-PCR): In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipeting steps and time, and is easy in handling, making it ideal for high throughput screening.
17. Reference Dye:
(Dye used in real-time experiments for normalization of the fluorescence signal of the reporter fluorophore. The reference dye fluoresces at a constant level during the reaction. ROX™ is commonly used as a reference dye.
18. Reference Gene:
Most common method of endogenous control for normalisation. Housekeeping genes are normally used, but they must validate over all the conditions to make sure their levels do not fluctuate over the course of the experiment.
| Gene |
Name |
Expression Level. |
| GAPDH |
Glyceraldehyde –3 – phosphate dehydrogenase |
High |
| ACTB |
β-Actin |
High |
| B2M |
β-2-microglobulin |
High |
| RRN18 |
18S ribosomal subunit |
Very High |
| UBC |
Ubiquitin C |
High |
| TBP |
TATAA-Box binding protein |
Low |
| HPRT1 |
Hypoxanthine-guanine phosphoribosyltransferase |
Medium |
| YMHAZ |
Tyrosine 3 / tryptophan 5-monooxygenase activation protein |
Medium |
19. Relative Quantitation:
To analyze changes in gene expression in a given sample relative to another reference samples (such as an untreated control sample).
20. Reporter Dye:
fluorophore): The fluorescent dye used to monitor PCR product accumulation in a real-time PCR experiment. This can be attached to a probe or free in solution (e.g. SYBR® Green I).
21. RT-PCR:
(Reverse Transcription PCR): This is used to amplify, isolate or identify a known sequence from a cell or tissues RNA library. Essentially normal PCR preceded by transcription by reverse transcriptase (to convert the RNA to cDNA) this is widely used in expression mapping, determining when and where certain genes are expressed.
22. Rn:
Normalized reporter signal, calculated by dividing the reporter signal by the passive reference signal (Rox or Fluorescein).
23. ΔRn:
Change in reporter signal as compared to baseline.
24. Sensitivity of Detection:
The level at which a given assay is able to detect low copy numbers. This is important when working with samples that have low expression levels.
25. Standard Curve:
The real-time PCR standard curve is a correlation plot generated by running a series of standards of known template concentration and then plotting the known starting quantities against the measured Ct values. The range of concentrations run should span the expected unknown concentration range. e.g.
R2: Measure of how well the points lie on the line (ideally ≥ 0.985)
M: Slop of the curve (ideally a 2x dilution gives an M of 1 and a 10x dilution gives an M of 3.32)
B: Indication of sensitivity (number of cycles required to say there is no target in the reaction)
Efficiency: Should be 90%-110% (M: 0.9-1.1 for a 2x dilution and 3.1-3.6 for a 10x dilution)
26. Threshold Cycle (Ct):
The PCR cycle at which fluorescence measured by the instrument is determined to be at a statistically significant level above the background signal (see amplification plot). The threshold cycle is inversely proportional to the log of the initial copy number.
27. Tm:
The midpoint of the melt phase, at which the rate of change in fluorescence is greatest, defines the temperature of melting (Tm) of the particular DNA fragment under analysis.
28. Two-Step PCR:
(See RT PCR) In a two-step reaction the reverse transcription reaction and the real-time PCR reaction are done in separate tubes. The cDNA can be used for more than one real-time PCR reaction and can be archived, eliminating the need to continually isolate RNA.
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