[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Messenger ribonucleic acid (mRNA) isolation from tomato seeds was performed with the PolyATtract System 1000 and was evaluated in two-step Real Time PCR using the fluoresence dye Sybr Green. The proposed isolation method resulted in cDNA production with Sybr Green 11-fold higher fluorescence intensity, having 87% lower standard deviation in the recorded fluorescence signals, and PCR efficiency improved by 21% in comparison to mRNA samples obtained with the manufacturer's protocol. The method for the assessment of the mRNA isolation procedure and the proposed mathematical analysis of the data are applicable to gene identification and expression studies for biotechnological applications.