New tools for efficient production of esiRNA and evaluation of their functionality

Abstract
In this study the functionality of endoribonuclease-prepared small interfering RNA (esiRNA) produced using the recently launched PowerCut™ Dicer (Finnzymes) was tested and compared to esiRNA produced using other commercially available enzymes. Double-stranded RNA was first produced with Replicator™ RNAi Kit (Finnzymes), which provides higher yield and specificity than methods based on hybridization of two separate RNA strands. Then, the produced dsRNA was cleaved to esiRNA molecules using three different dsRNA-cleaving enzymes. The RNAi experiments were carried out by transfecting HeLA cells constitutively expressing enhanced green fluorescent protein (eGFP) with various amounts (20, 10, 5 or 1 nM) of esiRNA targeting eGFP mRNAs. Targets and reference genes were first validated with DyNAmo™ Flash SYBR® Green qPCR kit and DyNAmo™ cDNA Synthesis Kit (FinnZymes) for qPCR-based relative gene expression analysis. In addition to eGFP, the targets included IFIT1, PKR and TLR3 for studying possible interferon responses. Twenty-three candidate primer pairs for reference genes were tested for their specificity and efficiency in qPCR, and ten out of those were selected for further analysis with geNorm. Based on geNorm results, two candidates were selected as reference genes for gene expression analysis. When compared to other enzymes, the cleavage efficiency of PowerCut Dicer appeared to be higher and the cleaved products more uniform in size. The results from gene expression analysis indicated that the silencing activity of esiRNAs produced by different enzymes was effective. No differences were found in the expression of the targets related to interferon response. The functionality studies have previously been evaluated with a VICTOR2 (PerkinElmer) multilabel counter, which measures the fluorescence of the eGFP expressed in HeLa cells. Good concordance between the eGFP gene expression data and fluorescense results was observed. The good yield and specificity of dsRNA produced with Replicator RNAi kit combined with the superior cleavage efficiency of PowerCut Dicer makes this combination a valuable tool for siRNA production.