Generic normalization method for real-time PCR. Application for the analysis of the mannanase gene expressed in germinating tomato seed.
A generic sample normalization method applicable in relative comparison of mRNAs quantified with real-time polymerase chain reaction (PCR) is proposed. The method was applied in samples obtained from tomato seeds after osmopriming and aging treatments and from untreated seeds at early imbibition stage, when seeds had not completed germination. Normalization in sample variations was accomplished by detecting synthetic DNA sequences tailing cDNA after second strand reverse transcription synthesis, while the use of the common normalizer GAPDH proved unreliable. Results, obtained from the new method and having a standard error less than 10%, verified the expression profile of a germination-specific mannanase gene that was closely recorded at different time intervals in relation to seed germination.