High Throughput Sample Preparation for RNAi Studies and Expression Profiling
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).
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.
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.
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 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.|