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. 2006:8:1-10.
doi: 10.1251/bpo114. Epub 2006 Jan 25.

Rapid and simple comparison of messenger RNA levels using real-time PCR

Andrée-Anne Dussault  1 , Marc Pouliot
Affiliations

Rapid and simple comparison of messenger RNA levels using real-time PCR

Andrée-Anne Dussault et al. Biol Proced Online. 2006.

Abstract

Real-time polymerase chain reaction (PCR) constitutes a significant improvement over traditional end-point PCR, as it allows the quantification of starting amounts of nucleic acid templates, in real-time. However, quantification requires validation through numerous internal controls and standard curves. We describe in this paper a simple protocol which uses real-time PCR to compare mRNA levels of a gene of interest between different experimental conditions. Comparative real-time PCR can be a relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set of experiments can be assessed in a single run. Thus, in addition to providing a comparative profile for the expression of a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species.

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Figures

Fig. 1
Fig. 1. The Rotor-Gene 3000 apparatus.
Fig. 2
Fig. 2. Illustration of a typical run using the Rotor-gene Analysis Software.
Fig. 3
Fig. 3. Typical real-time PCR result.
Fig. 4
Fig. 4. Example of a Melt® result confirming specificity of the results.
Fig. 5
Fig. 5. Example of a Melt® result showing non-specific amplification.
Fig. 6
Fig. 6. UV-equipped Plexiglas hood is used in order to prevent sample contamination.
Fig. 7
Fig. 7. Optimization of MgCl2 concentration in samples.
Fig. 8
Fig. 8. MgCl2: Melt® results.
Fig. 9
Fig. 9. Optimization of SYBR® Green concentration in samples.
Fig. 10
Fig. 10. SYBR® Green: Melt® results.
Fig. 11
Fig. 11. Linearity and efficiency test: GAPDH.
Fig. 12
Fig. 12. Linearity and efficiency test: COX-2.
Fig. 13
Fig. 13. Typical real-time PCR results obtained for GAPDH, COX-1 and COX-2, in saline- and LPS-stimulated leukocytes.
Fig. 14
Fig. 14. Comparative real-time PCR results expressed in fold increase, for COX-1 and COX-2, TNF-α, MIP-1α and MIP-1β in leukocytes stimulated with LPS.
Results represent the mean ± SEM from n=3 distinct experiments.
See this image and copyright information in PMC

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