Gene Expression Analysis in Arxula adeninivorans: A Nested Quantitative Real Time PCR Approach

Abstract

During the preparation of RNA traces of genomic DNA are usually co-isolated which might influence downstream applications. We tested several protocols, commercial kits and DNA hydrolysis procedures to remove the DNA contamination and found them to be insufficient. This can raise problems when it comes to gene expression analysis especially when working with intronless genes. Hence, we used a nested quantitative real time PCR approach to avoid amplification from genomic DNA by the use of an artificial anchor sequence introduced at the cDNA synthesis stage. This anchor sequence cannot be found in the genome of A. adeninivorans and a first round of amplification using a gene specific oligo in combination with an oligo for the anchor generates fragments which can emerge only from the cDNA. The second PCR step with nested oligos for the gene of interest and the anchor, respectively, significantly increases gene specificity which is crucial particularly when analysing the gene expression status among highly conserved members of a gene family. This second round of amplification represents the actual quantitative real time PCR assay.