Materials and Methods
Materials
? Bead Ruptor Elite (PN 19-040E)
? 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)
Methods
Cell Culture and Virus Growth
Human coronavirus 229e (HCoV-229e) was added at a MOI of 1.4 to 60% confluent MRC-5 (lung fibroblast) cells, 48 hours after plating. The flask was maintained with DMEM infused with 5% heat inactivated FBS and 1% L-Glutamine, incubated at 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 μL of supernatant was added to 300 μL of RLB buffer (from Omni Universal RNA Purification Kit, PN 26-010V) in either an 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, 1 x 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 homogenization step was completed, the remainder of the extraction was carried out per the manufacturer’s instructions for the OMNI Universal RNA Purification Kit with one exception: 100% EtOH was substituted for the 70% EtOH called for in step 7 of the kit’s protocol. RNA was eluted from the spin column using DPEC water, allowing an on-column reaction/dissolution time 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 gene was targeted with forward primer 5’-AGGCGCAAGAATTCAGAACCAGAG-3’ and reverse primer 5’-AGCAGGACTCTGATTACGAGAAAG-3’. 1 μL of extracted RNA was added, for a total reaction volume of 20 μL using the proportions of primers, RNA, SYBER, RT, and DPEC laid out in the New England Biologics Luna RT-qPCR Kit. The reaction was 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. The efficacy of the 4 homogenization methods for processing these samples was evaluated using Cq values (Figure 1) and confirmed with a 2% agarose gel (Figure 2). The data demonstrates successful extraction of viral RNA from cell culture supernatant using the OMNI Universal RNA Purification Kit. Bead Ruptor Elite homogenization increased extracted RNA yield in comparison to traditional vortexer, as demonstrated by lower Cq values in samples processed using bead milling homogenization versus vortexing. Increased band intensity during amplicon visualization is seen from homogenization using the Bead Ruptor Elite, both with and without 0.1 mm ceramic bead media, in comparison to the bands representing a vortexer, both with and without 0.1 mm ceramic bead media. These results were confirmed via 2% agarose visualization of amplicons as shown in Figure 2 and with quantified Cq values. In negative control replicates, N gene amplification on non-infected MRC-5 culture supernatant, the late rise was attributed to primer dimer formation in these samples.