Effect of T‐DNA insertions on mRNA transcript copy numbers upstream and downstream of the insertion site in Arabidopsis thaliana explored by surface enhanced Raman spectroscopy

Abstract

We report the effect of a T-DNA insertion on the expression level of mRNA transcripts of the TWISTED DWARF 1 (TWD1) gene upstream and downstream of the T-DNA insertion site in Arabidopsis. A novel approach based on surface-enhanced Raman spectroscopy (SERS) was developed to detect and quantify the effect of a T-DNA insertion on mRNA transcript accumulation at 5'- and 3'-ends of the TWD1 gene. A T-DNA insertion mutant in the TWD1 gene (twd1-2) was chosen to test the sensitivity and the feasibility of the approach. The null mutant of the FK506-like immunophilin protein TWD1 in Arabidopsis shows severe dwarfism and strong disoriented growth of plant organs. A spontaneous arising suppressor allele of twd1-2 called twd-sup displayed an intermediate phenotype between wild type and the knockout phenotype of twd1-2. Both twd1 mutant alleles have identical DNA sequences at the TWD1 locus including the T-DNA insertion in the fourth intron of the TWD1 gene but they show clear variability in the mutant phenotype. We present here the development and application of SERS-based mRNA detection and quantification using the expression of the TWD1 gene in wild type and both mutant alleles. The hallmarks of our SERS approach are a robust and fast assay to detect up to 0.10 fm of target molecules including the ability to omit in vitro transcription and amplification steps after RNA isolation. Instead we perform direct quantification of RNA molecules. This enables us to detect and quantify rare RNA molecules at high levels of precision and sensitivity.