High Throughput Sample Preparation for RNAi Studies and Expression Profiling

Introduction

The demand for robust high throughput RNA isolation and amplification protocols has increased very rapidly since RNAi and microarray technologies have gained wide application. We present: 1) high throughput siRNA synthesis, both chemically and enzymatically; 2) a comparison of various high throughput RNA isolation methods; and 3) a fully automated RNA amplification method for microarray analysis. We are working towards a systematic solution for both siRNA reagents and sample preparation for target discovery/validation.





dsRNA Synthesis for RNAi

Ambion offers genomewide pre-designed siRNAs using the proprietary design algorithm 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 amplification

The protocol is routinely used at Ambion for siRNA evaluation.

Figure 1. RNAqueous-MAG Performance and Application. Panel A shows high integrity and quality of isolated RNA; Panel B shows the RNA linear recovery; Panel C shows no cross contamination and efficiency in DNA removal. Panel D shows its application in siRNA evaluation (Human umbilical vein endothelial cells were transfected and mRNA was isolated 24 hr after transfection and quantified by qRT-PCR with SYBG).

*yield may vary with growth conditions.

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.

Figure 2. RNAqueous-96 Filter Plate Based RNA Isolation. K562 cells were used. Panel A shows the high quality of isolated RNA. Panel B shows the RNA linear recovery exemplified by GAPDH mRNA quantified by qRT-PCR.

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 bioanalyzer.

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 bioanalyzer. High quality aRNA was evident by the high yield and consistent size distribution with a maximum ~1500 nt.