qiaamp viral rna mini kit  (Qiagen)

Journal: International Journal of Nanomedicine

Article Title: Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology

doi: 10.2147/IJN.S191784

Figure Lengend Snippet: Quantitative analysis of the viral gene of rotavirus adsorbed onto the antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and then incubated with antibody-integrated magnetic beads. After incubation, the following fractions were obtained: 1) diluted rotavirus sample before incubation with the beads (BF), 2) bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), 3) bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), 4) supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), 5) supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and 6) total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). Viral genomic RNA was subsequently extracted from the above fractions using a QIAamp Viral RNA mini kit and subjected to RT-reaction. The resultant cDNA was analyzed by real-time PCR using primers for the rotavirus VP7 gene as described in Materials and methods. The value of the TL sample was taken as 100%. Abbreviations: MNBs, magnetic nanobeads; RT, reverse transcription.

Article Snippet: Viral genomic RNA was subsequently extracted from the above fractions using a QIAamp Viral RNA mini kit and subjected to a RT-reaction.

Techniques: Infection, Incubation, Magnetic Beads, Real-time Polymerase Chain Reaction

Journal: International Journal of Nanomedicine

Article Title: Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology

doi: 10.2147/IJN.S191784

Figure Lengend Snippet: Detection of viral RNA of rotavirus adsorbed onto antibody-integrated MNBs. Notes: Rotavirus-infected cell lysate (10 µL) was diluted with PBS (500 µL) and then incubated with antibody-integrated magnetic beads. After incubation, the following fractions were obtained: 1) diluted rotavirus sample before incubation with the beads (BF), 2) bead fraction after incubation with anti-rotavirus antibody-integrated MNBs (RV-BD), 3) bead fraction after incubation with anti-dengue virus antibody-integrated MNBs (DV-BD), 4) supernatant fraction after incubation with the anti-rotavirus antibody-integrated MNBs (RV-SP), 5) supernatant fraction after incubation with the anti-dengue virus antibody-integrated MNBs (DV-SP), and 6) total sample containing the same quantity of rotavirus as in 10 µL of rotavirus-infected cell lysate (total fraction, TL). Viral genomic RNA was subsequently extracted from the above fractions using a QIAamp Viral RNA mini kit and subjected to a RT-reaction. Rotavirus viral protein 7 (VP7) gene (552 bp) in the cDNA was amplified by PCR as described in Materials and methods. PCR products were analyzed by agarose gel electrophoresis (1.2% gel). The identity of the amplified products was confirmed by DNA sequencing. The left-hand lane is size marker (M), which includes DNA of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, and 1,500 bp. The position of the 552 bp band for VP7 is indicated by an arrow. The NC comprised a water sample (no rotavirus) that was subjected to RT-PCR. Abbreviations: MNBs, magnetic nanobeads; NC, negative control; RT, reverse transcription.

Article Snippet: Viral genomic RNA was subsequently extracted from the above fractions using a QIAamp Viral RNA mini kit and subjected to a RT-reaction.

Techniques: Infection, Incubation, Magnetic Beads, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, DNA Sequencing, Marker, Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Journal of Virology

Article Title: Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

doi: 10.1128/JVI.01832-15

Figure Lengend Snippet: Effects of V mutants on viral RNA synthesis. Truncated V proteins inhibited PIV5 RNA replication. BSR T7 cells were transfected as described in Materials and Methods. Encapsidated PIV5 minigenome RNA was purified with NP antibody, and a primer that binds to antigenomic RNA in the trailer region was added to the reverse transcription reaction mixture. Then, the same amount of RNA from the reverse transcription reaction was added to the PCR mixture as the template, and a pair of primers that anneal to the C terminus of Renilla luciferase and the PIV5 trailer region, respectively, was also added to the PCR mixture. The expected PCR products are indicated by MG and were approximately 250 bp. DNA ladder, a 100-bp DNA size marker (the bands of the ladder at 200 bp are indicated). (A) Detection of minigenome RNA using RT-PCR. Purified RNA without RT was used in the PCR mixture to demonstrate the lack of plasmid DNA contamination, and the corresponding RNA with RT was used in the PCR mixture. All samples were amplified for 30 cycles. (B) Detection of minigenome RNA using RT-PCR and different numbers of PCR cycles. Purified RNAs were reverse transcribed and then amplified for 15, 20, 25, and 30 cycles. No MG, cells transfected with all minigenome components except the plasmid carrying the minigenome; Pos Control, a minigenome system without plasmid carrying V or the V mutants; instead, the same expression plasmid containing enhanced GFP was used to maintain the total amount of transfected plasmid DNA constant. This gel is representative of those from three experiments. (C) Analysis of PCR products. The intensity of the PCR products was quantified using ImageQuant software. The intensity of cells transfected with all minigenome components except the plasmid carrying the minigenome was set equal to 0 in PCRs with 25 and 30 cycles; the intensity of the positive control was set to 100 in PCRs with 25 and 30 cycles; the relative percentage of the V, V120, or V21-222 PCR band intensity is the ratio of the V, V120, or V21-222 band intensity to that of the positive control in 25 and 30 cycles. The average was calculated from three independent experiments. Error bars represent standards deviation of the means. *, P

Article Snippet: The lysate was incubated with anti-NP214 antibody and recombinant protein G-Sepharose 4B beads at 4°C for 3 h. The beads were washed with PBS 3 times, and NP-encapsidated RNA was processed with a mini-viral RNA kit (Qiagen) and then treated with RNase-free DNase (Qiagen) for 3 h at 37°C to eliminate potential plasmid DNA contamination.

Techniques: Transfection, Purification, Polymerase Chain Reaction, Luciferase, Marker, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Amplification, Expressing, Software, Positive Control

Journal: Transfusion

Article Title: Antibody‐enhanced hepatitis E virus nanofiltration during the manufacture of human immunoglobulin, et al. Antibody‐enhanced hepatitis E virus nanofiltration during the manufacture of human immunoglobulin

doi: 10.1111/trf.16014

Figure Lengend Snippet: Characterization of untreated, S/D‐treated and S/D and protease–treated HEV. Buoyant densities of, A, wild‐type HEV from human plasma, B, rHEV, and C, wild‐type HEV from porcine feces without any further treatment (untreated), following S/D treatment (S/D treated), or after S/D and protease (pronase E) treatment. Virus was resolved in isopycnic gradients and RNA was detected by RT qPCR. LE particles have a density of 1.06 to 1.10 g/cm 3 , virions of IM band at 1.15 to 1.17 g/cm 3 , whereas NLE virions have a density of 1.19 to 1.22 g/cm 3

Article Snippet: 2.4 Detection of HEV RNAHEV RNA was extracted from volumes of 120 μL with a viral RNA isolation kit (QIAamp; Qiagen, Hilden, Germany).

Techniques: Quantitative RT-PCR