Samples found positive for IAV using the high-throughput rtPCR system, were selected for RNA extraction. The extraction was carried out for the selected nasal swabs using the QIAcube extraction robot (QIAGEN) and the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions as described previously [16 (link)]. RNA was eluted in 60 µL RNase-free water and stored at −80 °C. Detection of IAV in the extracted RNA was performed in the Rotor-Gene Q (QIAGEN) PCR platform using a previously published rtPCR assay targeting the matrix gene of IAV [15 (link),44 (link)].
Qiacube extraction robot
Manufactured by Qiagen
Sourced in United States, Germany
The QIAcube is an automated sample preparation instrument designed for the extraction and purification of nucleic acids (DNA and RNA) from a variety of sample types. The core function of the QIAcube is to automate the manual steps of the nucleic acid extraction process, providing consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Rotor gene q, by Qiagen (2 mentions)
Qiacube, by Qiagen (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
Stepone system, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Kapa sybr fast qpcr master mix 2x kit, by Roche (1 mentions)
Rapidout dna removal kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using qiacube extraction robot
1
Influenza A Virus Detection Protocol
2
Mosquito Capture and Identification Protocol
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Anopheles mosquitoes were caught in the Bijagós as part of ongoing studies undertaken by our group [17 , 18 ]. The mosquitoes were caught by using indoor CDC Miniature Light Traps (model 512; John W. Hock, Gainesville, FL) and previously described methodology [19 (link)]. All of the An. gambiae s.l. caught were identified using previously described morphological keys [20 (link)], killed using acetone, and stored dry using the same method as described above. The temperature at the time of preservation was 29 ± 3 °C and the relative humidity 80 ± 11%. Mosquitoes were then transported to a central laboratory where 350 randomly selected mosquitoes from across the archipelago were rehydrated and dissected. The ovaries were then scored by the first assessor. Photographs of the ovaries were taken using a microscope camera (Brunel Eyecam Plus; Brunel Microscopes, Chippenham, Wiltshire, UK) and then sent to two additional assessors for independent scoring by each. All of the assessors were blind to each other’s scores.
Confirmatory PCR–restriction fragment length polymorphism was performed on a subset of 45 samples at the Medical Research Council Unit The Gambia at LSHTM [16 (link)]. DNA was extracted by using a QIAcube extraction robot (QIAcube; QIAgen, Venlo, the Netherlands) in accordance with the manufacturer’s protocol.
Confirmatory PCR–restriction fragment length polymorphism was performed on a subset of 45 samples at the Medical Research Council Unit The Gambia at LSHTM [16 (link)]. DNA was extracted by using a QIAcube extraction robot (QIAcube; QIAgen, Venlo, the Netherlands) in accordance with the manufacturer’s protocol.
3
Molecular Detection of Gill Pathogens
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DNA was extracted from gill samples using the DNEasyKit (Qiagen®) on a QiaCube extraction robot (Qiagen®). To confirm the presence of P. perurans, gill samples of all fish were analysed using the qPCR assay as described by Downes et al. [33 ]. Gills of all naïve fish as well as the last two fish from each tank sampled at 21 and 35 dphe were also analysed for the presence of 'Candidatus' Branchiomonas cysticola (Bacteria), Desmozoon lepeophtherii (Microsporidia), Salmon Gill Pox Virus (SGPV) using the qPCR assays in Nylund et al [35 (link)], Mitchell et al. [36 (link)], and Gjessing et al. [15 (link)], respectively. For all qPCR analyses, samples were considered positive if the Cq-values were below the cut-off-values provided in the respective publications.
4
Total RNA Extraction from Blood Samples
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Total RNA was extracted from PAXgene Blood RNA tubes using the Qiagen PreAnalytiX PAXgene Blood miRNA Kit (Qiagen, Valencia, CA). The extraction protocol was performed either manually or with the Qiagen QIAcube extraction robot according to the company’s standard operating procedure.
5
RNA Extraction from PAXgene Tubes
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Total RNA was extracted from PAXgene ™ Blood RNA tubes using the Qiagen PreAnalytiX PAXgene Blood miRNA Kit (Qiagen, Valencia, CA). The extraction protocol was performed either manually or with the Qiagen QIAcube extraction robot according to the company’s standard operating procedure. Extracted RNA samples with RIN > 7 and concentration > =25 μg/ul were sequenced.
6
Transcriptome Analysis of Wound Tissues
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Total RNA extraction was performed as described by Nguyen et al. [26 (link)]. Briefly, each biopsy sample was transferred to 5 mL of TRIzol (Life Technologies, Carlsbad, CA, USA) and homogenized using the T25 ULTRATURRAX at 20,000 rpm (IKA, Staufen, Germany). RNA was extracted with 1 mL chloroform. The RNA was purified using an RNEasy Mini-Kit (Qiagen, Germantown, MD, USA) with a QIAcube extraction robot (Qiagen, Germantown, MD, USA). Genomic DNA was removed by treatment with DNAse I (Qiagen, Germantown, MD, USA). The RNA was quantified by NanoDrop One (Thermo Scientific, Karlsruhe, Germany)
Quantitative RT-PCR was performed as described by Karna et al. [27 (link)]. Briefly, quantitative real-time PCR was performed using the SYBR green master mix (Bio-Rad, Hercules, CA, USA) with specified primers (Supplemental Table S1 ) and was analyzed using the StepOne System (Applied Biosystems, Waltham, MA, USA). Expression of mRNA in wound tissues was determined by 2−ΔΔCt method and normalized by metal regulatory transcription factor 1 as the housekeeping gene. Assay was performed in technical triplicates.
Quantitative RT-PCR was performed as described by Karna et al. [27 (link)]. Briefly, quantitative real-time PCR was performed using the SYBR green master mix (Bio-Rad, Hercules, CA, USA) with specified primers (
7
RNA Extraction and RT-qPCR Analysis
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