SiRNA is widely used for analyzing gene function and target validation in vitro and in recent years has been successfully extended to in vivo research. Integrated DNA Technologies (IDT) synthesizes siRNAs for both in vitro and in vivo applications. Researchers planning in vivo work should be aware that potentially toxic substances can be introduced during oligonucleotide synthesis and purification (HPLC) processes, which must be removed to prevent erroneous results and/or the death of laboratory animals.
Elements of proper manufacturing protocols for siRNA intended for in vivo use that help eliminate toxic substances include:
• Adding trace EDTA in HPLC buffers to remove heavy metal cations.
• Using only biocompatible buffers and salts such as sodium phosphate and sodium chloride for HPLC.
• Maintaining an endotoxin-free environment.
The Endotoxin Problem
The classic endotoxin is lipopolysaccharide (LPS), a natural product present in the outer membrane of the cell wall of Gram negative bacteria. Toxic LPS is liberated whenever these bacteria die. If present as a contaminant in reagents used in vivo, it can cause severe inflammatory responses and/or death of the animals. In mammals LPS binds to serum proteins and interacts with various receptors on monocytes, macrophages, and endothelial cells. This eventually triggers cytokine production, complement activation, and coagulation cascades.
The majority of endotoxins are liberated when bacteria are lysed. These molecules are particularly difficult to degrade, being composed of a complicated sugar chain and lipid A. Interestingly, the major source of endotoxin contamination in manufacturing is water—in particular, sterile water.
The following guidelines help reduce the chance of LPS contamination of siRNAs intended for in vivo use:
• Follow standardized laboratory protocols for all stages of production.
• Work in a clean environment.
• Wear disposable gloves and other barriers.
• Maintain and clean the water purification system routinely.
• Autoclave all glassware at 180°C.
• Use high purity molecular biology grade salts for buffer preparation.
• Replace sterile water and buffers frequently to minimize potential for bacterial colonization.
• Use disposable sterile plasticware when possible.
• Use air filters to maintain an aseptic environment during ultrafiltration.
• Sterile filter the annealed duplex prior to lyophilization; release vacuum using an in-line sterile air filter to prevent bacterial ingress.
• Verify absence of endotoxins by QC testing the final product. We employ an external laboratory with a validated Limulus amebocyte lysate (LAL) test. "Pass" level should not exceed 2 EU/mg.
Using these protocols, detectable endotoxin levels for IDT's "in vivo clean siRNAs" are usually in the range of 0.01 to 0.1 EU/mg. This high level of care is needed to produce high quality reagents that are suitable for critical in vivo research and preclinical studies.