Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 19)

| 13 SsoAdvanced ™ Universal Probes Supermix Instruction Manual | 13
Tips for Success
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Pipet a minimum of 5 µl for each sample. This ensures greater precision and a smaller standard
deviation for technical replicates. If the samples are too concentrated, simply dilute accordingly.
Use a calibrated pipet of the appropriate volume range and never plunge the tip more than several
millimeters below the surface of the sample. Pipet slowly and use the pipet tip demarcations to
visualize accuracy
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Prepare individual master mixes for each sample by combining the real-time PCR supermix,
nuclease-free water, and primers along with the template and mix thoroughly. Then, pipet 20 µl
into the respective wells on the plate
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A tenfold dilution series is recommended to cover the most logs of dynamic range; however,
depending on the expression level of the gene(s) evaluated and the total template amount
available, this can be reduced to a fivefold dilution series
Linearity
Calculate the R 2 statistic for each standard curve using the qPCR analysis software; the R 2
should be ≥–0.980. However, if the R 2 is <0.980, remove outliers. If there are too many outliers,
then reevaluate the experiment to determine the cause of the lower R 2 value.
Dynamic Range
Determine the general trend of the slope where linearity (R 2 ) and efficiency are within acceptable
ranges, as specified above.
Sensitivity
Determine the lowest concentration of the serial dilution where replicate reproducibility is high
and the R
2 of the standard curve is ≥0.980.
Specificity
For probe-based assays, achieving optimal specificity requires observing a single band (PCR
product) following a gel analysis.
Efficiency
Calculate efficiency using the software or the following equation:
E =10
[–1/m] –1. A PCR efficiency from 90–110% (slope values from –3.6 to –3.1) is preferred.
Fig. 9. Reference gene has a PCR efficiency of 97.59% (–3.381) and six logs of
dynamic range. The target gene has a PCR efficiency of 99.17% (–3.342) with six logs of
dynamic range. Subtracting the slope values, 3.381 – 3.342 = 0.039, which is <0.1.
30
2 3 4 5
Log Starting Quantity
Cq
6 7 8
25
20
15