Quantitative transcript analysis in plants: improved first-strand cDNA synthesis.
The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived alpha-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 alpha-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.
Xiao
Nai-Zhong
NZ
School of Life Sciences, Northeast Normal University, Changchun 130024, China.
Ba
Lei
L
Holm
Preben Bach
PB
Wang
Xing-Zhi
XZ
Bowra
Steve
S
D015139:Blotting, Southern; D018076:DNA, Complementary / Q000096:biosynthesis*; D018744:DNA, Plant / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D029921:Trifolium / Q000235:genetics; D014404:Tubulin / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D014404:Tubulin