Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing

Abstract

Chitosan, a well known natural cationic polysaccharide, has been successfully implemented in vitro and in vivo as a nonviral delivery system for both plasmid DNA and siRNA. While using chitosan/siRNA polyplexes to knock down specific targets, we have underestimated the effect of nucleic acids binding to chitosan when extracting RNA for subsequent quantitative PCR evaluation of silencing. In vitro transfection using chitosan/siRNA-based polyplexes reveals a very poor recovery of total RNA especially when using low cell numbers in 96 well plates. Here, we describe a method that dramatically enhances RNA extraction from chitosan/siRNA-treated cells by using an enzymatic treatment with a type III chitosanase. We show that chitosanase treatment prior to RNA extraction greatly enhances the yield and the integrity of extracted RNA. This method will therefore eliminate the bias associated with lower RNA yield and integrity when quantifying gene silencing of chitosan-based systems using quantitative real time PCR.

Keywords: DPP-IV gene silencing; RIN; chitosan; chitosanase; qPCR; siRNA.

Figure 1

FACS analysis of chitosan/DPP-IV_ODN polyplexes uptake in HepG2 cell line. Uptake of 5′-6FAM labeled DPP-IV_ODN in chitosanase treated and untreated cells 24 hours post transfection. (A) Transfection efficiency was calculated as the percentage of 5′FAM-DPP-IV_ODN labelled cells. (B) The relative amount of internalized DPP-IV_ODN was determined using flow cytometry and is expressed as the median of fluorescence intensity (FI) of the 6FAM-DPP-IV_ODN positive cells. Data points represent the mean values ± SD, n = 3.*P < 0.05; **P < 0.01.

Figure 2

Confocal imaging of polyplexes uptake. Confocal microscopy images of HepG2 live cells 24 hours post transfection with chitosan/DPP-IV_ODN polyplexes (N/P = 5). Chitosan 92–10 (DDA, MW) was labeled with Rhodamine (red), the DPP-IV_ODN with 6FAM at the 5′extremity (green), and the cell membranes were stained prior to imaging with cell mask (blue). Membrane staining was performed to differentiate between internalized and membrane bound polyplexes.

Figure 3

Effect of Streptomyces griseus chitosanase on yield and integrity of total RNA extraction. Total RNA extraction was performed on HepG2 cells transfected with 10 pmol and 50 pmol of nanoparticles siRNA/chitosan at three different N/P ratios indicated by the formulation code 92-10-5, 92-10-10, or 92-10-20 (DDA, MW, N/P). Chitosanase was resuspended in DMEM pH 6.5 and directly applied to cells at a final concentration of 6.12 mU/μg of chitosan. Total RNA was extracted from chitosan transfected cell treated with or without chitosanase. The different extractions were compared to control Dharmafect™ 1 transfected cells and nontransfected (NT) cells. (nt) = nucleotide, L = standard ladder, the green band is a lower marker, which allows sample alignment and permits comparison for RIN calculation. RIN = RNA integrity number, is an algorithm-based numbering system that calculates-RNA integrity with 10 being the most intact and 1 being fully degraded.

Figure 4

Polyacrylamide gel electrophoresis of chitosan/DPP-IV_ODN polyplexes bearing different DDAs and N/P ratios, treated with or without Streptomyces griseus chitosanase. a) chitosan migration. b) ODN migration. Lanes 1 to 4 correspond to chitosan/DPP-IV_ODN directly incubated with chitosanase for 60 minutes at 37°C. Chitosan digestion allows the ODN release. Lanes 5 to 8 correspond to chitosan/DPP-IV_ODN incubated at the similar conditions without chitosanase. Faster chitosan migration was observed when comparing lanes 5 and 6 due to different MW of theses formulations. Increased band intensity (lanes 6–8) results from greater amounts of chitosan at higher N/P ratios.

Figure 5

Total RNA extraction from HepG2 transfected cells with 10 pmol siRNA. Following transfection, cells were treated with chitosanase for: 30 min and 60 min in cell medium and for 60 min in lysis buffer.