Production of a Human Genome-wide RNAi Library
The method uses long, double-stranded RNA molecules that are cleaved with Dicer or bacterial RNaseIII in vitro into overlapping siRNA molecules, which can then be introduced into mammalian cells to degrade the desired mRNA specifically while avoiding the cell’s antiviral defense mechanism. The starting material is a collection of 15,500 E. coli bacterial clones containing a genome-wide cDNA library (RZPD German Resource Center for Genome Research), which covers to the greatest possible extent all known and predicted human genes (Figure 1).
For each individual gene, the cDNA insertion fragments from the plasmids are amplified in 96-well plates using PCR with T7 polymerase promoters attached on both sides. The T7 polymerase reads off mRNA on both sides from these promoters and this is then hybridized to form double-stranded RNA. After adding recombinant RNaseIII from E. coli, the double-stranded RNA is cleaved into short overlapping siRNA fragments that contain the highly active effector molecules in the pool, in addition to the less active or completely inactive molecules.
This pool is purified through columns and, finally, the esiRNA concentrations for all esiRNAs are measured and adjusted to the same concentrations into new microtiter plates (normalization).
Gel-based quality-control steps are included at all important stages in order to verify the lengths of the fragments, purity, and concentration. All data obtained are fed into a database and all steps of the operation are monitored with a laboratory information management system. So far, a library has been obtained with over 14,000 molecule mixtures, available in the same concentrations in 384-well microtiter plates, in order to switch off a corresponding number of human genes in cultured cells and to investigate the effect on biological processes. Meanwhile, for certain parameters, an optimized human library has been produced, along with a genome-wide library for mouse.
The fundamental precondition for a well-adjusted and usable library is high-precision aliquoting and, subsequently, fully integrated measurement in terms of both hardware and software. For these reasons, a Freedom EVO Workstation with 8-channel liquid handling (LiHa) needles (Tecan) is used for the final step—the standardization of the library.
An integrated 96-channel Te-MO pipetting system with optional disposable pipette tips or a 96-channel steel needle head is used for rapid precision aliquoting, for the UV measurement of the esiRNA concentration. The Teflon-coated steel needles have the advantage that practically no serious retention of negatively charged siRNA occurs, as it does on the plastic surfaces of conventional interchangeable tips that usually have differing degrees of static charge and significantly greater surface roughness. Appropriate washing and sterilizing protocols were developed in order to avoid transferring substances onto the next plate or contaminating it with bacteria or fungi.
The workstation includes a fully integrated GENios Plus photometer (Tecan) to conduct the measurements at 260 and 280 nm, so that both the concentration and the purity can be determined by means of the quotient from the two measurements. For high throughput, the samples are aliquoted into 384-well plates (Corning) specially manufactured for this application. These plates have practically no absorption in the UV range and are less fragile than quartz cuvettes.
The best measurements are obtained when the samples are thoroughly mixed in the diluting medium by ‘sandwich’ pipetting (14 µL of diluting medium, 2 µL of sample, 14 µL of diluting medium) and by means of a separate mixing step. The concentration and purity measurements are exported automatically as a file in XML format straight into our LIMS at the TDS. Complete statistical analysis is carried out using this TDS-LIMS software and gel images are also imported into the TDS-LIMS. Samples of inadequate quality (no PCR band, PCR double-bands, incomplete RNaseIII digestion, etc.) or a too-low concentration are excluded.
The 8-needle LiHa arm is used to assemble the standardized library. Firstly, all eight needles can introduce the diluting medium, independently of each other and according to the instructions given by the LIMS, and then add the appropriate quantities of the esiRNA mixture as a summand, resulting in equal final volumes of the various esiRNAs in the same concentrations. Samples are then taken from this newly assembled library in 384-well plates, checked on a gel, and measured again in the UV spectrophotometer. The standard deviations after standardization lie within a range of less than 20% if effective use is made of the sample quantities (2 µL).