Comparison of standard exponential and linear techniques to amplify small cDNA samples for microarrays.

BACKGROUND: The need to perform microarray experiments with small amounts of tissue has led to the development of several protocols for amplifying the target transcripts. The use of different amplification protocols could affect the comparability of microarray experiments. RESULTS: Here we compare expression data from Pinus taeda cDNA microarrays using transcripts amplified either exponentially by PCR or linearly by T7 transcription. The amplified transcripts vary significantly in estimated length, GC content and expression depending on amplification technique. Amplification by T7 RNA polymerase gives transcripts with a greater range of lengths, greater estimated mean length, and greater variation of expression levels, but lower average GC content, than those from PCR amplification. For genes with significantly higher expression after T7 transcription than after PCR, the transcripts were 27% longer and had about 2 percentage units lower GC content. The correlation of expression intensities between technical repeats was high for both methods (R2 = 0.98) whereas the correlation of expression intensities using the different methods was considerably lower (R2 = 0.52). Correlation of expression intensities between amplified and unamplified transcripts were intermediate (R2 = 0.68-0.77). CONCLUSION: Amplification with T7 transcription better reflects the variation of the unamplified transcriptome than PCR based methods owing to the better representation of long transcripts. If transcripts of particular interest are known to have high GC content and are of limited length, however, PCR-based methods may be preferable.

Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR).

An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90-99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.