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

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.

Fig. 1. The Rotor-Gene 3000 apparatus.

Fig. 2. Illustration of a typical run using the Rotor-gene Analysis Software.

Fig. 3. Typical real-time PCR result.

Fig. 4. Example of a Melt® result confirming specificity of the results.

Fig. 5. Example of a Melt® result showing non-specific amplification.

Fig. 6. UV-equipped Plexiglas hood is used in order to prevent sample contamination.

Fig. 7. Optimization of MgCl2 concentration in samples.

Fig. 8. MgCl2: Melt® results.

Fig. 9. Optimization of SYBR® Green concentration in samples.

Fig. 10. SYBR® Green: Melt® results.

Fig. 11. Linearity and efficiency test: GAPDH.

Fig. 12. Linearity and efficiency test: COX-2.

Fig. 13. Typical real-time PCR results obtained for GAPDH, COX-1 and COX-2, in saline- and LPS-stimulated leukocytes.

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.