Molecular analyses of transgenic lines expressing pri-smiRNA(CHS). (A) Quantitative analysis of the relative transcript levels of the smiRNA(CHS) precursor by qRT-PCR experiments (RNA from wild type seedlings gave no signal) (B) RNA extracted from transgenic pri-smiRNA(CHS) lines numbered 1-11 was used for small RNA Northern blots to detect smiRNA(CHS) production. RNA from wild type (wt) and chs knockout (tt4) plants served as control. The upper panel shows signals obtained with the smiRNA(CHS) probe, the signals in the lower panel were obtained using a probe for U6snRNA as loading control. Size marker (M): 21 nt-long RNA oligonucleotide. (C) Summary of validation experiments using RLM-5'RACE experiments. The smiRNA(CHS):CHS mRNA hybrid is shown. Arrows and numbers refer to the positions and relative abundance of 5'RACE products analyzed. (D) Quantitative analysis of the CHS mRNA target by qRT-PCR experiments. Data in (A, D) were normalized to the highest values. The same transgenic lines were also analyzed in Figure 3.