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ISSN 1672-9145                                                Acta Biochim Biophys Sin 2005, 37(6):  429–434                                                  CN 31-1940/Q

Quantitative Transcript Analysis in Plants: Improved First-strand cDNA Synthesis

Nai-Zhong XIAO1, Lei BA1, Preben Bach HOLM2, Xing-Zhi WANG1*, and Steve BOWRA2*

 

1 School of Life Sciences, Northeast Normal University, Changchun 130024, China;

2 Department of Plant Biology, Research Centre Flakkebjerg, Danish Institute of Agricultural Sciences, DK-4200 Slagelse, Denmark

 

Abstract        The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional­ analysis and quantification. Using a plant-derived a-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved­ the quantity and quality of a-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.

 

Key words        cDNA; real-time PCR; RT-PCR; transcript analysis

 

 

 

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Received: December 14, 2004        Accepted: April 13, 2005

*Corresponding authors:

Xing-Zhi WANG: Tel, 86-431-5099769; E-mail, [email protected]

Steve BOWRA: Tel, +33-467617562; E-mail, [email protected]