Zymo rna clean concentrator tm 5 kit

RNA and DNA were extracted from gill tissue simultaneously, as previously described [19 (link)]. Briefly, tissue was lysed in TRIzol Reagent (Thermo Scientific, Waltham, MA, USA), and DNA and RNA phases were separated using chloroform. RNA was treated with DNAse I (Ambion, Austin, TX, USA) according to the RNA Clean & Concentrator-25 kit protocol (Zymo Research, Irvine, CA, USA). The concentration of RNA and DNA was measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific), and RNA integrity (RIN > 7.5; mean, 8.5) was assessed by a 2200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). In addition, we manually isolated 25 cysts from infected gills and extracted RNA using a Zymo RNA Clean & Concentrator TM-5 kit (Zymo Research) method (RIN = 7.1).
The infection severity of M. bejeranoi in fish gills was evaluated by qPCR, as previously described [19 (link)]. Briefly, specific primers targeted to amplify the M. bejeranoi small subunit ribosomal RNA gene (SSU rDNA), along with primers for Tilapia β-actin as a normalizer, were used on the extracted DNA. The computed qPCR relative quantity (RQ) was denoted as the relative infection severity index. From each sampling time point, three RNA replicates with similar mean RQ values (T0, 1.35 ± 0.21; T10, 7.65 ± 2.61; T20, 54 ± 8.84) were sent for sequencing.

RNA was extracted from human plasma samples (140 µL) using the QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) as per manufacturer's instructions. Mosquitoes were pooled per 50 individuals or less and homogenized with a vortex in ZR bashing bead lysis tubes containing 1 mL DNA/RNA shield. RNA was subsequently extracted from 200 µL homogenate according to the protocol of the Quick-DNA/RNA^TM Pathogen Miniprep Kit (Zymo Research, Germany). RNA from phocine distemper virus (PDV) was added to all samples as an internal RNA extraction and PCR inhibition control [16 (link)]. A CHIKV-specific RT-qPCR was then performed with 5 µL RNA in a 25 µL reaction using the iTaq Universal Probes One-Step Kit from Bio-Rad by amplifying a 77 bp part of the nonstructural protein 1 (NSP-1) gene with primers and probes (S Table 1) detecting the African and Asian CHIKV strains as previously described [17 (link)]. Cycling conditions were 10 min at 50°C, a denaturation step of 5 min at 95°C, followed by 50 cycles of 10 s at 95°C and 30 s at 60°C. A PDV RT-qPCR was run in parallel (S table 1). RNA samples with Ct-value >30 were concentrated to 10 µL using the Zymo RNA Clean & Concentrator^TM 5 kit (Zymo Research) prior to sequencing.