A significant challenge to large scale expression analysis and profiling projects is sample preparation. For example, manual sample preparation using Ambion's Amino Allyl MessageAmp aRNA Amplification Kit is typically limited to 1020 aRNA preparations over a two-day period, or approximately 2040 samples per week. The rate limiting step for increasing sample quantity is the labor-intensive column purification of cDNA and aRNA. Therefore the automation of mRNA preparation hinges on a robust and dependable method for purification. Whereas the standard column purification method requires extensive manual intervention, Ambion's MEGAclear magnetic bead purification technology is ideal for automation. The incorporation of a magnetic particle purification step after the cDNA synthesis step and then again after the in vitro transcription (IVT) step, allows aRNA sample preparation to be reformatted into a standard, automation-friendly 96 well plate.
GeneXP has validated and scaled up the automation of Ambion's Amino Allyl MessageAmp aRNA Amplification and 96 well MEGAclear purification system. The development of precise liquid-handling protocols allows maximization of purity and yields by careful control of RNA binding to and elution from magnetic beads. Consequently, it is now possible to amplify and purify diverse samples on multiple 96 well plates in the same two-day period that previously produced only 1020 aRNA preparations. This constitutes a greater than 10-fold improvement in throughput with high purity and high amplification yields, and with weekly throughput rising to 1,200+ samples per week on standard robots.
Figure 1 demonstrates the yield (Panel A) and purity (Panel B) of eight replicates of 2 g Promyelocytic Leukemia, Human Liver, and Universal Human Reference total RNA samples that were amplified and purified on two plates, for a total of 48 different samples. Samples were prepared as described by the Amino Allyl MessageAmp aRNA Amplification Kit protocol modified for robotic dispensation of reagents for the cDNA synthesis and amplification steps. Purification was conducted with magnetic beads for capture and elution of cDNA and aRNA.
Figure 1. Average Yield and Purity of aRNA Synthesized on a High Throughput Platform. (A) Eight 2 g replicates of three different total RNA samples (Ambion #7836 HL60 Promyelocytic Leukemia, Ambion #7960 Human Liver, Stratagene #740000 Universal Human Reference total RNA) were amplified and purified on two plates, for a total of 48 different samples. Samples were prepared as described by the Amino Allyl MessageAmp aRNA Kit protocol modified for robotic distribution of reagents for the cDNA synthesis and amplification steps. Purification was conducted with magnetic beads for capture and elution of cDNA and aRNA. (B) Yield and purity (A260:A280 absorbance ratio) were measured on a Tecan Genios Multi-Detection Microplate Reader using a 384 well BD Falcon Microtest UV-Vis microplate.
Similar results were also obtained using 1 g of starting material and typical yields from 1 g of the four different total RNA samples (brain, liver, HL60, and Universal Human Reference RNA) averaged 2040 g of aRNA, with purity equivalent to that obtained with 2 g. These results suggest that this automated process consistently yields a 500-fold or greater increase in aRNA product from total RNA sample.
Samples prepared by automation were evaluated by hybridization on a BGA plate, with pre- and post-hybridization processing performed with a robotic liquid handler. Figure 2 demonstrates representative hybridization results: reproducible gene-expression patterns, low background, and high signal-to-noise ratios.
Figure 2. Consistent, Reproducible Hybridization.HL60 aRNA (1 g/well; prepared with the automated methods described earlier) was hybridized in 96 wells of GeneXP Bioscience's BGA plate containing identical arrays of 50mer oligonucleotides designed to human target genes. Hybridizations were performed overnight in 80 l hybridization buffer using fragmented amino Allyl Cy5 aRNA (1 g) prepared from HL60 total RNA. Plates were processed using a Biomek FX (Beckman Coulter). Arrays were imaged using the Tecan LS200 scanner. The inset shows the hybridization results for four adjacent wells of the 96 BioGrid Array plate.
GeneXP has had excellent results incorporating Ambion's Amino Allyl MessageAmp aRNA Amplification Kit into its automated, high-throughput gene expression BGA platform. Expert tailoring of robot motion to the unique requirements of the kit and cleanup with magnetic particles were instrumental in developing a reproducible high-throughput process. Automation of the Amino Allyl MessageAmp aRNA Amplification Kit has enabled GeneXP to offer customers fast, affordable, and reliable high-throughput gene expression studies.
Amino Allyl MessageAmp aRNA