Competitive RT-PCR Strategy for Quantitative Evaluation -5
| 3. Characterization of the method precision and repeatability.a. Ensemble PCRs in the same conditions established before using quantities of target in the range of your expected measurements with different dilutions of competitor with replicas for each point. In our experiment, we ensemble:• 104 molecules of T with 5 × 103, 104, 5 × 104, 7.5 × 104, and 105molecules of C, respectively, with three replicas for each point.• 105 molecules of T with 104, 5 × 104, 7.5 × 104, 105, 5 × 105, and 7.5 × 105 molecules of C, respectively, with three replicas for each point. This experiment was repeated three times in different days to assay the inter-experiment variability.• 1.5 × 105 molecules of T with the same molecule quantities of C as the previous experiment with three replicas for each point.• 2 × 105 molecules of T with 103, 104, 5 × 104, 105, 5 × 105, and 106molecules of C, respectively, with three replicas for each point.• 106 molecules of T with 5 × 105, 106, 5 × 106, and 107 of C, respectively, with three replicas for each point.b. Electrophoresis in 2% agarose gel in TA 1× (0.04 M Tris–acetate, 0.001 M EDTA, pH 7) of the amplification products of each tube.c. Take digital images of the gel.d. Densitometry analysis of competitor and target bands in each lane using Image J Program (Version 1.33) as was described in point 1d of this section.e. Make lineal regressions for each experiment plotting log of the ratio between the area measurements of C and T versus log of the C molecule numbers. Determine the equation of each regression with its respective R2.f. Determine the x value (C molecule number) when y = 0. This condition occurs when the area measurements of C and T are equal because C/T = 1 and the log1 = 0. Then, x value is equal to the calculated T molecular number in each experiment.g. Plot T molecule numbers expected versus observed and determine the correlation coefficient (r). We used the test of Pearson correlation. This determination represents a measure of the precision of our method in the range of the T quantities assayed.h. Calculate the variation coefficients (CVs) intra-experiment using the replicas inside experiments and inter-experiments. These CV values characterize the repeatability of our method in the range of the T quantities assayed. | |
| III. Competitive RT-PCR Materials• RNAgents: Total RNA Isolation System, Promega Z5110• Competitor RNA generated in the Protocol I• Gene-specific primers to amplify target and competitor sequence.• Ready-to-Go RT-PCR Beads, Amersham Biosciences, 27-9259-01• Reagents and equipment to do DNA electrophoresis.• Digital camera Olympus C7070 Wide Zoom• Image J program (Version 1.33). Methods1. Isolate total RNA of eight different tissues (brain, muscle, heart, liver, gonads, stomach, spleen, and intestine) from three tilapias (O. niloticus). Total RNA mini-preparations starting with 50 mg of each tissue were performed using RNAgents. Total RNA Isolation System, Promega. (see table 1 of the TB087).2. Ensemble reactions of RT-PCR using Ready-to-Go RT-PCR Beads, Amersham Biosciences. We used 2 µl of each preparation of total tissue-specific RNA with at least three known different quantities of competitor RNA and random primers to the RT reactions and 0.2 µM of each specific primer to the PCR.3. Electrophoresis in 2% agarose gel in TA 1× (0.04 M Tris–acetate, 0.001 M EDTA, pH 7) of the amplification products of each tube.4. Take digital images of the gel.5. Densitometry analysis of competitor and target bands in each lane using Image J Program (Version 1.33) as was described in point 1d of this section.6. Do lineal regressions for each experiment plotting log of the ratio between the area measurements of C and T versus log of the C molecule numbers. Determine the equation of each regression.7. Determine the x value (C molecule number) when y is equal to 0. In this condition, x value is equal to T molecule number in each RNA sample.8. Normalize this T molecule number in each RNA sample with the input of total RNA in the RT reaction. The total RNA input was determined by densitometry analysis of 28S subunit of ribosomal RNA in each sample using Image J program. We did an electrophoresis on 1.2% agarose denaturalizing gel of 2 µl of each total RNA preparation. The densitometry data of the bands corresponding to the 28S subunits measured with Image J program were converted to micrograms of total RNA using a reference RNA with known concentration. Then, the results were expressed as tiGHR I molecule numbers/µg RNAt.9. Average the tiGHR I molecule numbers/µg RNAt among the three tilapias to each tissue. These averages were compared by non-parametric Kruskal–Wallis test followed by Dunn multiple comparison test. |
Fig. 7 Densitometry analysis of competitor and target bands using Image J Program.a User-defined selection from 2% agarose gel digital image. b Image J plots of selected image.c The area measurements displayed in a Results window by the image J Program.
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