tailieunhanh - Báo cáo khoa học: 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. | iFEBS Journal Generic normalization method for real-time PCR Application for the analysis of the mannanase gene expressed in germinating tomato seed Dimitris Argyropoulos1 Charoula Psallida1 and Caroline G. Spyropoulos2 1 Institute of Biotechnology NationalAgriculturalResearch Foundation Athens Greece 2 Department of Botany Faculty of Biology University of Athens Greece Keywords gene expression mannanase normalization real-time polymerase chain reaction relative quantification Correspondence D. Argyropoulos Institute of Biotechnology NationalAgriculturalResearch Foundation S. Venizelou 1 141 23 Athens Greece Fax 211 7508893 Tel 210 2842676 or 211 7508894 E-mail biomol@ Received 26 October 2005 revised 9 December 2005 accepted 19 December 2005 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 mannan-ase gene that was closely recorded at different time intervals in relation to seed germination. doi Gene expression studies include quantification of mRNAs being produced under different conditions such as stress growth development and cell and tissue localization or as part of evaluation of the effects of gene transfection. A variety of techniques exist to quantify mRNAs and usually involve northern hybridization ribonuclease protection or real-time PCR assays 1 2 . In real-time PCR .