Stored surplus urine specimens from CMV IgG seropositive non-Hispanic white, non-Hispanic black, and Mexican American participants were tested for the occurrence of viral shedding.44 (link) Viral DNA was extracted with the QIAmp MinElute Media Kit and processed with the Qiacube automated extractor (both manufactured by Qiagen, Valencia, California). Detection of CMV DNA was performed by a real-time PCR assay that targeted the highly conserved immediate-early 2 exon 5 region using the MX 3005P Real-time PCR System (Agilent Technologies, New Castle, Delaware).45 (link) The lower limit of detection was 80 copies/ml, equivalent to 80 international units per milliliter (SC Dollard, written communication, May 2019). PCR testing was performed in duplicate for all specimens, with two positive results required for specimens to be reported as positive.44 (link) A summary of the CMV laboratory tests used in this analysis is provided in Table 1 of the Supplemental Digital Content; http://links.lww.com/EE/A90 .
Qiacube automated extractor
Manufactured by Qiagen
Sourced in Germany
The QIAcube is an automated sample preparation instrument that performs nucleic acid extraction and purification. It automates the complete sample-to-result workflow, reducing manual handling and increasing reproducibility.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna ffpe tissue kit, by Qiagen (4 mentions)
Therascreen egfr rgq pcr kit egfr ivd kit, by Qiagen (3 mentions)
Mx3005p real time pcr system, by Agilent Technologies (2 mentions)
Qiamp minelute media kit, by Qiagen (1 mentions)
Qiaamp dna formalin fixed paraffin embedded tissue kit, by Qiagen (1 mentions)
7500 fast system real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rhamp snp genotyping system, by Integrated DNA Technologies (1 mentions)
Dna blood mini kit, by Qiagen (1 mentions)
Qiaamp minelute media kit, by Qiagen (1 mentions)
Egfr ruo kit, by Qiagen (1 mentions)
Qiamp tissue elution buffer, by Qiagen (1 mentions)
Rnasy kit, by Qiagen (1 mentions)
Geneamp gold rna pcr core kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
9 protocols using qiacube automated extractor
1
Cytomegalovirus DNA Detection in Urine Specimens
2
Detecting EGFR Mutations in Lung Cancer
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Tumor tissues were obtained from the primary lung tumors or metastatic lesions of the patients for EGFR mutation analysis. Only tissue samples consisting of >80% tumor content, as evaluated via microscopy, were used for this purpose. For each sample, DNA was extracted using the QIAcube automated extractor (Qiagen, Hilden, Germany) with the QIAamp DNA Formalin-Fixed Paraffin-embedded (FFPE) Tissue Kit (Qiagen) and then eluted in ATE (QIAmp Tissue Elution) buffer (Qiagen) according to the manufacturer’s instructions. The EGFR PCR Kit (EGFR RUO Kit) and the Therascreen EGFR RGQ PCR Kit (EGFR IVD Kit, Qiagen, Manchester, UK) were then used in combination with the Scorpions and amplification-refractory mutation system (ARMS) technologies to detect the presence of EGFR mutations by real-time quantitative PCR.
3
FTO Gene Genotyping from Whole Blood
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Samples of 5 mL of whole blood were collected from the cubital vein and stored under refrigeration. Genomic DNA was extracted using the DNA Blood Mini Kit (Qiagen, CA, United States) using a QIA cube automated extractor (Qiagen, CA, United States), according to the manufacturer's recommendations. Then, they were stored in a freezer at −20 °C for an indeterminate period in order to avoid possible loss of material or contamination.
A NanoDrop spectrophotometer (Termo Scientific, CA, United States), was used for the quantification of the extracted DNA, according to the manufacturer's instructions.
The analysis of the rs9939609 SNP of the FTO gene was performed using the rhAmp™ SNP Genotyping System (Integrated DNA Technologies, IA, United States) assay on a 7500 Fast System real-time PCR system (Applied Biosystems, CA, United States). The reagents were purchased commercially and used according to the manufacturer's standards.
A NanoDrop spectrophotometer (Termo Scientific, CA, United States), was used for the quantification of the extracted DNA, according to the manufacturer's instructions.
The analysis of the rs9939609 SNP of the FTO gene was performed using the rhAmp™ SNP Genotyping System (Integrated DNA Technologies, IA, United States) assay on a 7500 Fast System real-time PCR system (Applied Biosystems, CA, United States). The reagents were purchased commercially and used according to the manufacturer's standards.
4
EGFR Mutation Analysis in Lung Cancer
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To perform the EGFR mutation analysis, tumor tissues were collected from the primary lung cancer or metastatic lesions, with tissue samples having a tumor content of more than 80% as determined using microscopy with hematoxylin and eosin staining being selected for the analysis. DNA was extracted using a QIAamp DNA FFPE tissue kit (Qiagen) eluted in ATE (QIAmp Tissue Elution) buffer in combination with the QIAcube automated extractor (Qiagen) according to the manufacturer's instructions. The EGFR PCR Kit (EGFR RUO Kit) and the therascreen EGFR RGQ PCR Kit (EGFR IVD Kit, Qiagen) were used to identify mutated EGFR DNA. These kits utilized a combination of Scorpions real‐time PCR technology and amplification‐refractory mutation system (ARMS) technology for the detection of the mutations with real‐time quantitative PCR.22
5
Urine-based CMV DNA Detection
6
Lung Cancer EGFR Mutation Analysis
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The tissue of primary or metastatic lung cancer was obtained for EGFR mutation analysis. Tissue sample consisting over 80% tumor content, as determined via microscopy with hematoxylin and eosin staining, were selected for the study. The QIAcube automated extractor (Qiagen, Hilden, Germany) with the QIAamp DNA FFPE tissue kit (Qiagen) eluted in ATE (QIAmp Tissue Elution) buffer (Qiagen) were used to extract DNA according to the manufacturer's instructions. The presence of EGFR mutations was determined using the EGFR PCR Kit (EGFR RUO Kit) and therascreen EGFR RGQ PCR Kit (EGFR IVD Kit). These kits combined Scorpions and the amplification-refractory mutation system (ARMS) technologies to detect the mutations using real-time quantitative PCR.
7
Molecular Detection of EGFR Mutations
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Tumor tissues were obtained from the primary lung mass for analysis. Tissue samples were selected if they had >80% tumor content, as examined with microscopy in hematoxylin and eosin staining. Subsequently, DNA was extracted through the QIAcube automated extractor (Qiagen) with the QIAamp DNA FFPE tissue kit (Qiagen), eluted in ATE (QIAmp Tissue Elution) buffer (Qiagen) according to the manufacturer's instructions. The EGFR polymerase chain reaction kit (EGFR RUO Kit) and Therascreen EGFR RGQ PCR Kit (EGFR IVD Kit, Qiagen) were used to detect EGFR mutations. Finally, scorpion and amplification‐refractory mutation system technologies were combined to disclose the mutations via real‐time quantitative polymerase chain reaction.14
8
Genomic DNA and RNA Extraction
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Genomic DNA from the patients, their parents, and healthy controls was isolated from peripheral blood using QIAamp DNA Mini Kit and the QIAcube automated extractor (QIAGEN, Hilden, Germany). DNA quality and concentration were measured using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE, USA), and it was stored at −20°C.
Ficoll gradient centrifugation was used to isolate mononuclear cells from peripheral blood from the patients and healthy controls, following the manufacturer's recommendations (Lymphoprep AXIS-SHIELD PoCAS). Once purified, mononuclear cells were lysed in RLT buffer and total RNA was isolated using the RNasy kit (QIAGEN), as recommended by the manufacturer. RNA quality and concentration were measured also with the NanoDrop ND-1000 Spectrophotometer. Total RNA was reversely transcribed in a final volume of 40 μl, following the manufacturer's guidelines (Geneamp Gold RNA PCR Core Kit, Applied Biosystems, Foster City, CA, USA), and immediately stored at −80°C.
Ficoll gradient centrifugation was used to isolate mononuclear cells from peripheral blood from the patients and healthy controls, following the manufacturer's recommendations (Lymphoprep AXIS-SHIELD PoCAS). Once purified, mononuclear cells were lysed in RLT buffer and total RNA was isolated using the RNasy kit (QIAGEN), as recommended by the manufacturer. RNA quality and concentration were measured also with the NanoDrop ND-1000 Spectrophotometer. Total RNA was reversely transcribed in a final volume of 40 μl, following the manufacturer's guidelines (Geneamp Gold RNA PCR Core Kit, Applied Biosystems, Foster City, CA, USA), and immediately stored at −80°C.
9
EGFR Mutation Detection in Lung Tumor
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Tumor tissues were obtained from the primary lung mass, metastatic lymph node, or pleural tumor for analysis. These samples were only selected if the tumor component qualified for the EGFR mutation assay based on prior microscopy examination with hematoxylin and eosin staining. Tumor DNA was extracted using a QIAcube automated extractor (Qiagen) with the QIAamp DNA FFPE Tissue Kit (Qiagen) and eluted in QIAmp Tissue Elution (ATE) buffer (Qiagen) according to the manufacturer's instructions. The EGFR RUO polymerase chain reaction (PCR) kit and Therascreen EGFR RGQ PCR Kit (EGFR IVD Kit; Qiagen) were used to detect EGFR mutations. Finally, scorpion and amplification‐refractory mutation system technologies were combined to identify the mutations via a real‐time quantitative PCR.15
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