High Throughput Sample Preparation for RNAi Studies and Expression Profiling

Xingwang Fang, Roy C Willis, Quoc Hoang, Michael A Siano, Sharmili Moturi and Kevin Kelnar
Ambion, Inc.• 2130 Woodward, Austin, TX 78744 • Phone: 512-651-0200 • Fax: 512-651-0201

dsRNA Synthesis for RNAi
Ambion offers genomewide pre-designed siRNAs using the proprietary design algorithmn from Cenix Biosciences. Over 200 siRNAs have been validated by analysis of mRNA levels after siRNA transfection. Validation has been defined as target mRNA reduced by more than 70%. We’ve also developed in vitro transcription kits based on Ambion’s patented high-yield MEGAscript® transcription technology to produce double stranded RNA (both short dsRNA for siRNA and long for RNAi in non-mammalian systems). As shown in Scheme 1, dsRNA is conveniently synthesized in a single reaction mixture that can be easily automated. In each 20 µl transcription reaction, the RNA yield is 50-100 µg for long dsRNA and 10-20 µg for short dsRNA, enough for hundreds of transfections.

RNA Isolation with Magnetic Beads
Solid phase RNA isolation with magnetic beads is very suitable for high throughput applications, and the process is easy to automate. The advantages of Ambion’s new RNAqueous®-MAG technology include:
• high purity, integrity and yield of RNA with high consistency
• only 1-1.5 hours required to process up to 4 plates in parallel
• RNA is eluted in 20-50 ml water for easy integration with qRT-PCR or mRNA amplication
The protocol is routinely used at Ambion for siRNA evaluation.

RNA Isolation with Filter Plate
High quality RNA can also be isolated in 96-well format using glass fiber filter plates. We have improved Ambion’s RNAqueous-96 technology to meet the ever rising standards. The new reagents and protocols are more user friendly and give higher RNA yield and consistency. RNA is typically eluted in 50-100 µl water, and processing of up to 4 plates in parallel takes about 1 hour.

Sample Preparation for Microarray Gene Profiling
Ambion’s MEGAscript® high yield transcription kit and MessageAmp™ kit are widely used for RNA labeling for Affymetrix GeneChips and other oligo arrays. Now we have streamlined the entire process of sample preparation for microarray analysis: RNA isolation --> cDNA synthesis --> in vitro Transcription --> aRNA labeling. Magnetic beads are used in all isolation/purification steps, enabling seamless processing. The whole process can be carried out on a robot integrated with a thermocycler and an orbital shaker.

Figure 3. Automatation-friendly Cleanup (bead-based) vs. Manual Cleanup (filter-based). The bead-based cleanup method enables streamlining of the whole procedure because of the negligible dead volume of beads. When filter plates are used for cleanup, cDNA has to be concentrated before using for IVT amplification and aRNA is very diluted.	Figure 4. Efficient Dye Labeling of Amino Allyl RNAs and Recovery of Dye-labeled aRNA of Various Sizes. Amino allyl RNAs were generated with the Amino Allyl MessageAmp Kit protocol using Century™-Plus Marker Template mix. The RNA mixture, before and after dye coupling, was analyzed on an RNA 6000 chip with Agilent bioanaylzer.

Figure 5. Reproducibility of Amplified RNA Yield from a Fully Automated Procedure in a 96-well Format. Sample input was 2 µg of Hela S3 total RNA per well. aRNA was quantified by A260 on a BioTek UV plate reader. Average cRNA yield is 132 ± 13 µg (C.V. <10%).	Figure 6. Size Profile of Amplified RNA. 12 randomly selected aRNA samples were run on a RNA 6000 chip with Agilent bioanaylzer. High quality aRNA was evident by the high yield and consistent size distribution with a maximum ~1500 nt.