MRC-5 (肺成纤维细胞)细胞中冠状病毒RNA检测方案(均质器)

APPLICATIONNOTE 202004The Homogenizer Company RNA Extraction from Human Coronavirus withOMNIUniversal RNA Purification Kit and Bead Ruptor Elite Rodney Nash， Ph.D.，Brandon Easparro， Caleb Proctor， Zachary Morehouse，OMNI International， Inc. Summary Extraction of viral RNA from tissue samples is a critical method used in confirming the presence of a suspected viralinfection. With viral infections on the rise globally，researchers and clinicians from all sectors have turned their focus toincreasing research on virally induced pathologies. In this protocol we have demonstrated that the OMNI Universal RNAkit can be used to extract viral RNA from human Coronavirus 229e (HCoV-229e) in the supernatant of infected humanlung fibroblast tissue culture flasks. Additionally， these samples were processed using the Bead Ruptor Elite， reducing the time needed for the initialhomogenization steps of RNA kit extractions by half. This semi-automated sample preparation step increased throughputof traditional spin column RNA extraction protocols allowing for processing up to 24 samples in 30 seconds， wheretraditional methods require up to 24 minutes for the same procedure. Use of the Bead Ruptor Elite for RNA kit processing resulted in increased viral RNA product yield as detected by RT-qPCR，suggesting that low concentration samples may be detectable. Through amplification of the nucleocapsid geneof HCoV-229e from tissue culture supernatant， we demonstrated an increased efficiency and efficacy of bead millhomogenization over vortexhomogenization when conducting viral RNA extractions using traditional spin column kits. Materials and Methods Materials ·Bead Ruptor Elite (PN19-040E) ·2mL Tube Carriage Kit (PN 19-010-310) ·Universal RNA Purification Kit (PN 26-010V)， includes： ·Universal Microbial Homogenizing Mix， Nuclease Free (PN 19-632)·2mL Reinforced Tubes with Screw Caps (PN 19-649) 30 RUPTORELITE OMNI Methods Cell Culture and Virus Growth Human coronavirus 229e (HCoV-229e) was added at a MOl of 1.4 to 60% confluent MRC-5 (lung fibroblast) cells， 48 hoursafter plating. The flask was maintained with DMEM infused with 5% heatinactivated FBS and 1% L-Glutamine， incubatedat 37 C with 5% CO2. The cell culture supernatant was harvested at 72 hours post infection when 70% CPE was observed. Viral RNA Extraction from Supernatant 300 pL of supernatant was added to 300 pL of RLB buffer (from Omni Universal RNA Purification Kit， PN 26-010V) in eitheran empty 2 mL homogenization tube (PN 19-649) or a prefilled Universal Microbial Homogenizing Mix tube (PN 19-632).Sample tubes were then homogenized by one of two methods： 1， using the Bead Ruptor Elite， 1x 30s cycle at 4.2 m/s；or 2， vortexing for 60s using a vortex mixer similar to the Vortex Mixer 24 (PN 28-101). After the initial homogenizationstep was completed， the remainder of the extraction was carried out per the manufacturer's instructions for the OMNIUniversal RNA Purification Kit with one exception： 100% EtOH was substituted for the 70% EtOH called for in step 7 ofthe kit’s protocol. RNA was eluted from the spin column using DPEC water， allowing an on-column reaction/dissolutiontime of 5 mins prior to centrifugation. HCoV-229e RT-qPCR HCoV-229e nucleocapsid gene (N gene) was selected as a target for RT-PCR from peer reviewed publications.The N geneWastargeted with forward primer5-AGGCGCAAGAATTCAGAACCAGAG-3' and reverse primer5'-AGCAGGACTCTGATTACGAGAAAG-3.1 pL of extracted RNA was added， for a total reaction volume of 20 pL using theproportions of primers， RNA， SYBER，RT， and DPEC laid out in the New England Biologics Luna RT-qPCR Kit. The reactionwas run for 44 cycles and the resulting amplicons were loaded into a 2% agarose gel for product visualization. Results RT-qPCR was completed on the supernatant of MRC-5 tissue culture flasks， 72 h.p.i. once 70% CPE was observed. Theefficacy of the 4 homogenization methods for processing these samples was evaluated using Cq values (Figure 1) andconfirmed with a 2% agarose gel (Figure 2). The data demonstrates successful extraction of viral RNA from cell culturesupernatant using the OMNI Universal RNA Purification Kit. Bead Ruptor Elite homogenization increased extracted RNAyield in comparison to traditional vortex homogenization， as demonstrated by lower Cq values in samples processedusing bead milling homogenization versus vortexing. Increased band intensity during amplicon visualization is seenfrom homogenization using the Bead Ruptor Elite， both with and without 0.1 mm ceramic bead media， in comparison tothe bands representing vortex homogenization， both with and without 0.1 mm ceramic bead media. These results wereconfirmed via 2% agarose visualization of amplicons as shown in Figure 2 and with quantified Cq values. In negativecontrol replicates， N gene amplification on non-infected MRC-5 culture supernatant， the late rise was attributed to primerdimer formation in these samples. Amplification Cycles Figure 1： RT-qPCR Cq values for HCoV-229e N gene amplification visualization Light blue： Bead Ruptor Elite homogenization in an empty 2 mL reinforced tube (PN 19-649) Orange： Vortexer homogenization using an empty 2 mL reinforced tube (PN 19-649) Dark blue： Bead Ruptor Elite homogenization in pre-filled Universal Microbial Homogenizing Tubes (2 mL reinforced tube pre-filled with 0.1 mm ceramic bead media， PN 19-632) Yellow： Vortexer homogenization using pre-filled Universal Microbial Homogenizing Tubes (PN 19-632). Green： Negative control replicates， N gene amplification on non-infected MRC-5 culture supernatant. Sample Cq Cq Mean Cq Standard Deviation Bead Ruptor Elite-19-649-Sample 1 11.08 11.61 0.48 Bead Ruptor Elite-19-649-Sample 2 11.72 Bead Ruptor Elite-19-649-Sample 3 12.03 Bead Ruptor Elite-19-632-Sample 1 13.61 13.86 0.22 Bead Ruptor Elite-19-632-Sample 2 13.95 Bead Ruptor Elite-19-632-Sample 3 14.02 Vortex Mixer 24-19-649-Sample 1 13.03 13.27 0.40 Vortex Mixer 24-19-649-Sample 2 13.05 Vortex Mixer 24-19-649-Sample3 13.73 Vortex Mixer 24-19-632-Sample1 12.92 13.02 Vortex Mixer24-19-632-Sample 2 13.16 Vortex Mixer 24-19-632-Sample3 12.97 Figure 1B： RT-qPCR results for HCoV-229E nucleocapsid gene showing the quantified Cqresults for each of the homogenization parameters tested in triplicate. 12345678910 11 12 13 1415 Figure 2：HCoV-229e N gene amplicon expression after RT-qPCRon a 2% agarose gel Lane 1： BioRad 100bp DNA ladder Lanes 2，3， 4： MRC-5 cells at 70% CPE supernatant viral RNA extractionusing vortex homogenization with empty 2mL tubes Lanes 5， 6，7： MRC-5 cells at 70% CPE supernatant viral RNAextraction using vortex homogenization with pre-filled UniversalMicrobial Homogenizing Mix Lanes 8，9， 10：MRC-5 cells at 70% CPE supernatant viral RNA extractionusing Bead Ruptor Elite agitation with empty 2 mL tubes Lanes 11， 12， 13： MRC-5 cells at 70% CPE supernatant viral RNAextraction using Bead Ruptor Elite homogenization with pre-filledUniversal Microbial Homogenizing Mix Lane 14：Empty x Bead Ruptor Elite Universal RNA Purification Bundle19-040V-Indudes ·Bead Ruptor Elite (PN 19-040E) ·2 mL Tube Carriage Kit (PN 19-010-310 ·26-010V Universal RNA Purification Kit26-010V 技科         近日，美国OMNI Inc公司联合多位科学家发表了Bead Ruptor 24 Elite多功能生物样品均质器用于抽提人冠状病毒RNA的技术方案。        此方案不仅详细阐述了如何进行人冠状病毒RNA抽提的方法步骤，并且对使用不同设备、耗材的抽提效果进行了详细对比。        实验结果证明，使用Bead Ruptor 24 Elite均质器可大大提高人冠状病毒RNA抽提率。