https://www.abbs.info
E-mail: [email protected]
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
—————–
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]