Data Analysis

Results obtained using real-time RT-PCR assays are significantly less variable than conventional RT-PCR

Cycle number

Fig. 2 Threshold cycle Ct. The threshold cycle is defined as the number of PCR cycles where the fluorescence generated from the amplification product first exceeds a baseline level. It depends on the sensitivity of the detection system and can vary significantly depending on assay-specific background levels. The two amplification plots have Ct values that differ by six cycles (i.e., represent an approximately 100-fold difference in template starting copy numbers).

Cycle number

Fig. 2 Threshold cycle Ct. The threshold cycle is defined as the number of PCR cycles where the fluorescence generated from the amplification product first exceeds a baseline level. It depends on the sensitivity of the detection system and can vary significantly depending on assay-specific background levels. The two amplification plots have Ct values that differ by six cycles (i.e., represent an approximately 100-fold difference in template starting copy numbers).

Fig. 3 Preparation of a standard curve. (A) Sense-strand amplicon-specific oligonucleotides are serially diluted from 1 x 108 to 10 copies, and their respective Ct values are recorded. (B) A plot of Ct against the log of the initial oligonucleotide copy number results in a straight line that is linear over at least seven orders of magnitude, and linear regression analysis permits the calculation of the ''absolute'' copy number of any unknown target relative to that standard curve.

Fig. 3 Preparation of a standard curve. (A) Sense-strand amplicon-specific oligonucleotides are serially diluted from 1 x 108 to 10 copies, and their respective Ct values are recorded. (B) A plot of Ct against the log of the initial oligonucleotide copy number results in a straight line that is linear over at least seven orders of magnitude, and linear regression analysis permits the calculation of the ''absolute'' copy number of any unknown target relative to that standard curve.

copy number is used to generate a standard curve by plotting the Ct values against the logarithm of the initial copy numbers (Fig. 3).[29] Its dynamic range must include the Ct values expected for the experimental RNA samples. The copy numbers of unknown samples can be calculated from the linear regression of that standard curve, with the slope providing the amplification efficiency. Standard curves can be constructed from PCR fragments, in vitro T7-transcribed RNA, single-stranded sense-strand oligodeoxyribonucleotides, or commercially available universal reference RNAs.[30]

Absolute quantification is most obviously used for quantifying tumor cells or infectious particles such as viruses or bacteria in body fluids, but it is also usefully applied to quantitate changes in mRNA levels. The accuracy of absolute quantification depends entirely on the accuracy of the standards. However, external standards cannot detect or compensate for inhibitors that may be present in the samples.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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