For HBV RNA Northern blot analysis, total cellular RNA or encapsidated pgRNA samples were resolved in a 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond-XL membrane (GE Healthcare). For DNA Southern blot analysis, HBV core DNA or cccDNA samples were resolved by electrophoresis in a 1.2% agarose gel and blotted onto a Hybond-XL membrane. Membranes were probed with either an [α-32P]UTP (3,000 Ci/mmol; PerkinElmer)-labeled plus-strand-specific (for Northern blot hybridization) or minus-strand-specific (for Southern blot hybridization) HBV riboprobe and exposed to a phosphorimager screen. Extracellular HBV DNA and RNA were copurified from the culture medium of induced HepAD38 cells by using a MinElute virus vacuum kit (Qiagen). Viral DNA was quantified using the above-mentioned qPCR method directly, and HBV RNA was detected by RT-qPCR with the same core region primer/probe set after the removal of DNA contamination by DNase I (Thermo Fisher) treatment.
Minelute virus vacuum kit
The MinElute virus vacuum kit is a laboratory equipment product designed for the extraction and purification of viral nucleic acids from a variety of sample types. It utilizes a silica-based membrane technology to efficiently capture and concentrate viral RNA or DNA, enabling their subsequent analysis or downstream applications.
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2 protocols using minelute virus vacuum kit
Comprehensive HBV Nucleic Acid Extraction
For HBV RNA Northern blot analysis, total cellular RNA or encapsidated pgRNA samples were resolved in a 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond-XL membrane (GE Healthcare). For DNA Southern blot analysis, HBV core DNA or cccDNA samples were resolved by electrophoresis in a 1.2% agarose gel and blotted onto a Hybond-XL membrane. Membranes were probed with either an [α-32P]UTP (3,000 Ci/mmol; PerkinElmer)-labeled plus-strand-specific (for Northern blot hybridization) or minus-strand-specific (for Southern blot hybridization) HBV riboprobe and exposed to a phosphorimager screen. Extracellular HBV DNA and RNA were copurified from the culture medium of induced HepAD38 cells by using a MinElute virus vacuum kit (Qiagen). Viral DNA was quantified using the above-mentioned qPCR method directly, and HBV RNA was detected by RT-qPCR with the same core region primer/probe set after the removal of DNA contamination by DNase I (Thermo Fisher) treatment.
Thyroid Tissue and Plasma DNA Isolation
For plasma DNA isolation, two 5 mL-aliquots of peripheral blood were collected in EDTA tubes, transported within one hour to the laboratory and centrifuged twice at 4℃ for 10 min (1600 rcf and 14000 rcf; or at 415 g or 1660 g). Plasma was aliquoted into 1.5 mL tubes after centrifugation, and stored at -80℃ until genetic analysis. DNA was extracted from 500 µL of plasma, using the MinElute Virus Vacuum Kit (Qiagen) and RNAse digestion to prevent RNA interference during assay reaction. Plasma DNA was extracted with the QIAamp DNA Blood Midi Kit (Qiagen) according to the manufacturer's instruction. The extracted DNA yields were similar in the respective tumor and plasma specimens.
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