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208 Bartlett
3.4. Calculation of Results
1. The results of a typical assay are illustrated in Fig. 1. After restriction digestion of the
co-amplified mutant and normal RI alpha, two bands are clearly visible, representing
products of 430 and 215 base pairs. If no normal cDNA is added, all the product is digested
to give only a 215-bp band. In the illustrated assay, mutant RI alpha cDNA is co-amplified
in a range of concentrations with known (100 or 10 pg) or unknown (patient sample)
concentrations of unmutated RI alpha cDNA. At high concentrations of mutant plasmid,
the lower band is the most intense. As the concentration of mutant cDNA is decreased,
the relative intensity of the lower 215 bp band decreases and that of the larger 430 bp
band increases. Where concentrations are equivalent, each product is produced at the
same intensity. Thereafter, the upper 430-bp band becomes more intense. The point of
equivalence of concentration is therefore represented by a crossover between the lower
and upper band intensities.
2. For each sample, the counts per minute determined by Cerenkov (1) counting for the
mutant and normal bands are plotted against the concentration of mutant plasmid added.
The point at which the two curves cross represents the point at which the concentration of
mutant and normal RI alpha are equivalent, thus allowing the concentration in unknown samples
to be determined from this crossover point (see Fig. 2). In this example, the counts for
the mutant and normal RI alpha bands from the PCR assay are plotted against the concentration
of mutant RI alpha added. The curves cross over at 10 pg, indicating a concentration
in the test sample of 10 pg RI alpha cDNA equivalent to 3.4 fmol RI alpha mRNA in
the sample.
3. The sensitivity of the assay was assessed using a range of cDNA concentrations from
34 fmol to 0.0034 attomol (10 –14 to 10 –21 mol, 30 cycles of PCR were used for this lower
limit). The sensitivity under these conditions was 0.002 attomol (2 × 10 –21 mol or approx
1000 copies of mRNA; see Note 12).
4. Intra and interassay variation for the quantitative PCR assay were determined as 8.0 and
14.3%, respectively (see Note 13).
5. Calculation of confidence intervals for results: As the co-efficients of variation are known
for each stage of the reverse transcription (RT)-PCR assay, we were able to calculate
the confidence intervals for sample concentrations determined by this assay technique.
Where samples are measured within the same assay this is calculated as follows (see
Note 13):
C imax = C o (1 + E i )(1 + V i ) and C imin = C o (1 – E i )(1 – V i )
Where C imax is the maximum estimated concentration and C imin is the minimum. C o is the
observed concentration, E i is the intra-assay variation for reverse transcriptase, and V i is
the intra-assay variation for PCR.
If necessary interassay confidence limits can be defined from the above values using
the following formula:
C bmax = C imax (1 + E b )(1 + V b ) and C bmin = C imin (1 – E b )(1 – V b )
Where C bmax is the maximum estimated concentration and C bmin is the minimum and E b
is the inter-assay variation for reverse transcriptase and V b is the intra-assay variation
for PCR.
3.5. Discussion
By assessing the variation at each step during the RT-PCR procedure, this method
defines the variation as a result of the reverse transcription and PCR steps and show that