1. Abstract Automated, High-Throughput Total and Viral RNA Purification Byung-in Lee 1, Seachol Oak 1, Sharon Khietala 2, Beate Schikora 2, and Karl H Hecker 1 1 Invitrogen Corporation Carlsbad, CA 2 California Animal Health and Food Safety Lab, University of California, Davis, Davis, CA Gene expression analyses are a key element in identifying target genes for drug development and molecular diagnostics. Reliable and reproducible analyses are dependant on the availability of high quality total RNA, preferably obtained by a high throughput, auto- mated purification process. Concert™ 96 RNA Purification System, which is based on glass microfiber filter technology, meets these quality and throughput requirements. The system uses a universal 96-well plate design that is compatible with most robotic platforms for the isolation of total RNA from a variety of sources such as bacteria, yeast, plant and tissue samples, mammalian cells, and whole blood. Total RNA can be reliably isolated from as few as 100 to as many as 5 x 10 5 mammalian cells with high well-to-well consistency, excellent total RNA recovery, and without well-to-well cross contamination as determined by RT-PCR. In an effort to expand the utility of total RNA isolation, the system was tested for its ability to co-purify viral RNA for downstream diagnostic uses. Tracheal and cloacal swab samples of healthy chicken were spiked with serial dilutions of a vaccine strain of the Exotic Newcastle Disease (END) prior to RNA isolation. The presence of viral RNA in the final RNA preparation was detected with high sensitivity by gene specific RT-PCR using TaqMan technology. Introduction Results Source Materials 1.35 4.40 7.46 9.49 2.37 (kb) 1324567 Various source samples, e.g.: E. coli, yeast, plant tissue, mouse & human cells, were processed in an automated format to purify total RNA. Total RNA purified with the Concert 96 RNA Purification Kit was of high integrity as evidenced by the sharpness of large rRNA transcripts in the RNA preparations obtained from various sources. Figure 1: Gel analysis of total RNA purified with the Concert 96 RNA Purification System Lane 1: 0.24 – 9.5 Kb RNA Ladder ( Invitrogen) Lane 2: 0.7 μg of total RNA purified from E.coli (1x10 8 cells) Lane 3: 0.8 μg of total RNA purified from S. cervisiae (1x10 8 cells) Lane 4: 1.0 μg of total RNA purified from lettuce leaves Lane 5: 1.1 μg of total RNA purified from mouse NIH3T3 (3.7x10 5 cells) Lane 6: 1.4 μg of total RNA purified from human 293H (3.7x10 5 cells) Lane 7: 1.0 μg of total RNA purified from human HeLa (3.7x10 5 cells) Extraction of Viral RNA The Concert 96 RNA Purification System is based on the binding of crude total and viral RNA to glass microfiber filters. Total and viral RNA bound to this silica-based matrix is subjected to procedures aimed at removing contaminants and eluted with RNase-free water in a final step. In order to support this high-throughput, automated format, all wash steps and the elution step are performed by vacuum filtration. DNA contamination of the RNA isolated can further be controlled and minimized by the introduction of an on-filter DNase digestion step during the purification procedure. The Concert 96 RNA Purification System is compatible with the majority of current robotic automation systems due to its universal plate design. The RNA purification procedure is optimized for automation and suitable for a variety of source samples. RNA purified from various source materials showed high purity, minimal genomic DNA contamination, high RNA integrity, reproducible yields, high column capacity, and no well-to-well cross contamination as determined by RT-PCR. Further- more, it is shown that the Concert 96 RNA Purification System can be used without modifications for viral RNA extraction/co-purification providing high quality viral RNA preparations for pathogen detection and diagnostic application. Exotic Newcastle Disease-negative (END-negative), 500-  l swab samples (tracheal and cloacal swabs in liquid medium) obtained from healthy, non-infected chicken and spiked with APMV-1 vaccine virus (National Veterinary Services Laboratories) were subjected to RNA isolation with the Concert 96 RNA Purification System. END- negative swap samples were spiked with a stock APMV-1 vaccine virus preparation to yield dilutions of the virus of 10 -2 – 10 -5 in order to mimic END-infection. Spiked samples were processed for viral RNA co-purification using the protocol for total RNA purification. 9-  l aliquots of the eluted RNA, elution volume 150  l, were tested for the presence of viral RNA by quantitative RT-PCR with virus- specific primers and probes using the Platinum ® Quantitative RT- PCR ThermoScript™ One-Step System from Invitrogen. Viral RNA from the vaccine strain of the END virus could be co-purified with host total RNA and detected for up to 100,000-fold dilutions. The efficiency of RNA purification combined with the assay’s sensitivity facilitates diagnostic and surveillance applications. Conclusion vaccine virus in swap (500  L) Ctvaccine virus only Ct no virus 1/100 1/1,000 1/10,000 1/100,000 50.0 26.5 29.3 36.7 46.2 no virus 1/100 50.0 28.4 Sensitivity – Minimum Cell Number Required The Concert 96 RNA Purification System was used successfully for the isolation of high-quality total RNA from as few as 100 HeLa cells in a fully automated, high-throughput format. Presence and quality of total RNA isolated was assessed by RT-PCR with a set of gene- specific LUX™ hSDHA primers. Only 5  l of a total elution volume of 140  l was used for RT-PCR corresponding to the reliable detection of hSDHA mRNA in the total RNA equivalent of only 4 HeLa cells. Temp ( o C) -d(RFU)/dT CF RFU Cycle Threshold analysis Melting temp analysis Consistent Well-to-Well Performance Total RNA was purified from 96 HeLa cell samples in parallel with the Concert 96 RNA Purification System. Q RT-PCR was performed with a gene specific LUX™ SDHA primer set while RNA concentrations were determined directly by RiboGreen ® RNA quantification. Average Ct for RNA isolated from 10 5 HeLa cells was 24.5  1.3 (SD) and for 10 4 HeLa cells 28.2  1.2 (SD). Total RNA yields obtained from 10 5 and 10 4 HeLa cells, respectively, were 1.45  0.17 ng/μl (SD) and 0.06  0.02 ng/μl (SD). The Concert™ 96 RNA Purification System represents a versatile, fast, and reliable kit for high-quality, sensitive, reproducible, auto- mated, and high-throughput isolation and purification of total RNA. Viral RNA can be efficiently co-purified with host total RNA for downstream viral detection and screening. 96 samples can be processed in parallel on a robotic workstation in less than one hour, which includes a 15-min DNase I digestion step. Genomic DNA Contamination w/ RT rxn w/o RT rxn Host genomic DNA (gDNA) is an unavoidable contaminant in all total RNA preparations. However, gDNA can be efficiently removed by DNase I treatment during or post RNA isolation. Here we employed an on-filter DNase I digestion step to remove contaminating gDNA. Quantitative PCR was performed on total RNA isolated using the on- filter DNase I digestion after reverse transcription (RT) and without RT to assess the extent of gDNA contamination. In order to achieve the latter, a primer set was used that indiscriminately amplifies gDNA and cDNA. The average cycle number (Ct) required for amplification after RT of the total RNA purified was 25 as compared to 39 Ct when RT was not performed. A 14 Ct difference translates into a residual gDNA contamination after DNase I digestion of less than ca. 0.01 %. Figure 2: Quantitative RT-PCR analysis of APMV-1 viral RNA extracted with the Concert 96 RNA Purification System Figure 3: Quantitative RT-PCR analysis of total RNA purified from A549 cells assessing host genomic DNA contamination Figure 4: Q RT-PCR with total RNA isolated from HeLa cells Blue: Total RNA isolated from 10,000 HeLa cells Red: Total RNA isolated from 1,000 HeLa cells Yellow: Total RNA isolated from 100 HeLa cells Green: No-cell control Figure 5: Well-to-well variation analysis of 96 RNA samples with RiboGreen and quantitative RT-PCR.