Prev Article：Molecular Diagnostic Knowledge - qPCR Terminology (Part 2)
14. Standards
A sample of known concentration used to construct a standard curve. To ensure the stability of the standards, the gene fragments are usually cloned into plasmids and used as standards.
15. Standard Curve
A standard of known concentration is diluted in a gradient (usually 5-6 dilution gradients), and the unknown sample and the gradient diluted standard are reacted in the same qPCR experiment. Based on the amplification curve, the Ct value of the diluted standard is used as the horizontal coordinate and the log value of the starting copy number is used as the vertical coordinate to construct. In theory, the amplification curves of diluted samples have a uniform spacing between them. 3.32.
16. Amplification Efficiency
The amplification efficiency is related to the slope of the standard curve, and the absolute value of the slope is the same as the spacing of the fluorescence curve. The amplification efficiency is calculated as E = 10-1/slope, and if the amplification efficiency is expressed as a percentage, E% = (E-1) × 100%. In general, amplification efficiency close to 100% is a sign of good reproducibility, however, in practice, the amplification efficiency should be between 90% and 105%. If the amplification efficiency is low, the possible reasons are improper primer design or unoptimized reaction conditions; if the amplification efficiency is greater than 100%, it may be due to the wrong dilution sample addition, or there is non-specific product amplification, such as primer dimer generation.
17. Internal Reference
A control sequence that is amplified as a target sequence in the same reaction and detected by double PCR with different probes.
18. Reference Gene
The expression levels of endogenous reference genes do not differ between samples, such as Housekeeping genes (HKGs), commonly used as GAPDH, β-actin, 18SrRNA and 28S rRNA.
19. Standard Sample
A reference sample used in relative quantification to determine the relative expression level of a gene by comparison with other samples.
20. Positive Control
A control with a known amount of template, usually used to check that the primers are working properly and that the reaction program is set up correctly.
21. Negative Control
Important negative controls include No template control (NTC) and No reverse transcriptase control (NRC).
NTC:
A control reaction containing all the necessary components for an amplification reaction except the template, which is usually replaced by water and is used to detect reagent contamination or contamination caused by exogenous DNA.
NRC:
One more system is prepared during reverse transcription without adding reverse transcriptase, and the rest of the components are added normally. The product obtained under the same reverse transcription process is used as a template to observe the amplification and for testing whether there is contamination of genomic DNA in cDNA.
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