High pure viral rna kit

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The High Pure Viral RNA Kit is a laboratory product designed for the rapid and efficient extraction of viral RNA from various sample types. It utilizes a silica-based purification method to capture and purify viral RNA, which can then be used for downstream applications such as reverse transcription and real-time PCR analysis.

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One step tb green primescript rt pcr kit 2, by Takara Bio (2 mentions) One step tb green primescript rt qpcr kit 2, by Takara Bio (1 mentions) Pcr purification combo kit, by Thermo Fisher Scientific (1 mentions) Primescript rt pcr kit, by Takara Bio (1 mentions)

4 protocols using high pure viral rna kit

SARS-CoV-2 RNA Extraction and RT-qPCR Detection

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200 μL of the supernatant was harvested from cells infected with SARS-CoV-2 and mixed with equal volume of lysis buffer provided in the high pure viral RNA kit (Invitrogen) and RNA was extracted according to the kit's instructions. The eluted RNA was subjected to RT-qPCR by using the One Step TB Green PrimeScript RT-qPCR Kit II (Takara) and specific primers targeting the SARS-CoV-2 nucleocapsid protein (NP):
Forward, 5′-TAATCAGACAAGGAACTGATTA-3′;
Reverse, 5′-CGAAGGTGTGACTTCCATG-3′.
RT-qPCR cycling conditions were 42 °C for 5 min, 95 °C for 10 s, and 40 cycles of 95 °C for 5 s, followed by 60 °C for 30 s.
All the SARS-CoV-2 based experiments were performed at the UCLA BSL3 facility.

Aliyari S.R., Ghaffari A.A., Pernet O., Parvatiyar K., Wang Y., Gerami H., Tong A.J., Vergnes L., Takallou A., Zhang A., Wei X., Chilin L.D., Wu Y., Semenkovich C.F., Reue K., Smale S.T., Lee B, & Cheng G. (2022). Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2. Acta Pharmaceutica Sinica. B, 12(4), 1624-1635.

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Quantifying SARS-CoV-2 Viral RNA in Cell Lines

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Huh7 and A549-hACE2 cells were infected with SARS-CoV-2 at an MOI of 0.01 for the indicated time. Then 200 μL of the supernatant culture was harvested for viral RNA extraction with the high pure viral RNA kit (Invitrogen) or virus titer evaluation. RNA was finally eluted with 30 μL of RNase-free water and used as the template for RT-PCR quantification. For qPCR analysis, the specific primers that target SARS-CoV-2 N gene were used: forward, 5′-TAATCAGACAAGGAACTGATTA-3′; reverse, 5′-CGAAGGTGTGACTTCCATG-3′. RT-qPCR was performed using One Step TB Green PrimeScript RT-PCR Kit II (Takara) with the following cycling conditions: 42 °C for 5 min, 95 °C for 10 s, and 40 cycles of 95 °C for 5 s, followed by 60 °C for 30 s.

Zhang S., Wang J, & Cheng G. (2021). Protease cleavage of RNF20 facilitates coronavirus replication via stabilization of SREBP1. Proceedings of the National Academy of Sciences of the United States of America, 118(37), e2107108118.

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SARS-CoV-2 Quantification in Vero-E6 Cells

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Vero-E6 cells were infected with SARS-CoV-2 at an MOI of 0.01 for 24 h or 48 h. Two hundred microliters of cell culture medium was harvested for viral RNA extraction with a High Pure Viral RNA kit (Invitrogen). RNA was eluted with 30 µl of RNase-free water and used as the template for RT–PCR quantification. For qPCR analysis, the following specific primers targeting the SARS-CoV-2 N gene were used: forward, 5′-TAATCAGACAAGGAACTGATTA-3′, and reverse, 5′-CGAAGGTGTGACTTCCATG-3′. RT–qPCR was performed using One Step TB Green PrimeScript RT–PCR Kit II (Takara) and the following cycling conditions: 42 °C for 5 min, 95 °C for 10 sec, and 40 cycles of 95 °C for 5 sec, followed by 60 °C for 30 sec.

Zhang S., Wang J., Wang L., Aliyari S, & Cheng G. (2022). SARS-CoV-2 virus NSP14 Impairs NRF2/HMOX1 activation by targeting Sirtuin 1. Cellular and Molecular Immunology, 19(8), 872-882.

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NDiV Viral RNA Detection and Sequencing

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Suspensions of cells showing CPE were processed for RNA extraction with the High Pure Viral RNA Kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. A reverse transcription-polymerase chain reaction (RT-PCR) method was used for NDiV-specific gene (RdRp) detection with the Prime Script RT-PCR Kit (TaKaRa Biotechnology, Dalian, China). The primer sequences were: forward primer PRd1 5′-TCACACAACCGCATGCTACGC-3′ and reverse primer PRd2 5′-GTGGTCACGGCCAGTGGTGT-3′ (targeting a 2394 bp fragment). The polymerase chain reaction (PCR) products were visualized on 2% agarose gels and extracted from the gels using the PCR Purification Combo Kit (Invitrogen). The PCR fragments were sequenced and compared to known RdRp gene sequences of NDiV in the National Center for Biotechnology Information (NCBI) GenBank database by BLASTN.

Zhou J., Jin Y., Chen Y., Li J., Zhang Q., Xie X., Gan L, & Liu Q. (2017). Complete Genomic and Ultrastructural Analysis of a Nam Dinh Virus Isolated from Culex pipiens quinquefasciatus in China*. Scientific Reports, 7, 271.

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