Mouse organs were homogenized in RNALater, and viral RNA was isolated using the QIAamp 96 virus QIAcube HT kit (Qiagen). Two microliters of RNA was used as a template for the amplification of selected genes by real-time quantitative PCR (qRT-PCR) using HiSxript II one-step qRT-PCR SYBR green kit (Vazyme). Average values from duplicates of each gene were used to calculate the viral genomic copies. The primers based on the SARS-CoV-2 S gene were RBD-qF1, 5′-CAATGGTTTAACAGGCACAGG -3′, and RBD-qR1, 5′-CTCAAGTGTCTGTGGATCACG -3′. For qRT-PCR, the 10-μL mix contained 5 μL 2× one-step SYBR green mix, 1.9 μL nuclease-free water, 0.5 μL one-step SYBR green enzyme mix, 0.2 μL 50× ROX reference dye 1, 0.2 μL of each primer (10 m M), and 2 μL template RNA. Amplification was performed at 50°C for 3 min, 95°C for 30 s followed by 40 cycles consisting of 95°C for 10 s and 60°C for 30 s, and a default melting curve step in a Step-One Plus real-time PCR machine (ABI).
Hisxript 2 one step qrt pcr sybr green kit
Manufactured by Vazyme
The HiSxript II one-step qRT-PCR SYBR green kit is a real-time reverse transcription PCR (qRT-PCR) reagent system designed for sensitive and accurate quantification of RNA targets. The kit combines reverse transcription and PCR amplification in a single reaction for streamlined workflow.
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4 protocols using hisxript 2 one step qrt pcr sybr green kit
1
SARS-CoV-2 S Gene Quantification
2
SARS-CoV-2 Viral RNA Quantification
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Mouse organs were homogenized in RNALater, and viral RNA was isolated using the QIAamp ® 96 Virus QIAcube ® HT kit (QIAGEN). Two microliters of RNA were used as a template for the amplification of selected genes by real-time quantitative PCR using HiSxript ® II One step qRT-PCR SYBR® Green Kit (Vazyme). Average values from duplicates of each gene were used to calculate the viral genomic copies. The primers based on the SARS-CoV-2 S gene were designed as: RBD-qF1: 5′- CAATGGTTTAACAGGCACAGG-3′; RBD-qR1: 5′- CTCAAGTGTCTGTGGATCACG-3′. The PCR system was as follows: the 10 μL qPCR reaction mix contained 5 μL 2 × One Step SYBR Green mix, 1.9 μL nuclease free water, 0.5 μL One Step SYBR Green Enzyme mix, 0.2 μL 50 × ROX Reference Dye 1, 0.2 μL of each primer (10 μM) and 2 μL template RNA. Amplification was performed as follows: 50°C for 3 min, 95°C for 30 s followed by 40 cycles consisting of 95°C for 10 s and 60°C for 30 s, and a default melting curve step in an Step-One Plus Real-time PCR machine (ABI) (Zhou et al., 2020 (link)).
3
SARS-CoV-2 RNA Quantification by qRT-PCR
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Mouse organs were homogenized in DMEM, and viral RNA was isolated using the QIAamp ® Viral RNA Mini Kit (QIAGEN). HiSxript ®II One step qRT–PCR SYBR® Green Kit (Vazyme) was used to amplify the selected genes by real-time quantitative PCR, and 2 μL of RNA were used as a template. Viral genomic copies were calculated by average values from duplicates of each gene. The primers were designed based on the SARS-CoV-2 S gene as follows: RBD-qF1: 5′-CAATGGTTTAACAGGCACAGG-3′; RBD-qR1: 5′-CTCAAGTGTCTGTGGATCACG-3′. The PCR system followed the protocol of the previous study using a Step-One Plus Real-time PCR machine (ABI)3 (link).
4
SARS-CoV-2 RNA Detection in Mice
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Mouse organs were homogenized in DMEM, and viral RNA in the samples was quantified by one-step qRT-PCR. Viral RNA was extracted using the QIAamp viral RNA minikit (Qiagen) and then used as the template for qRT-PCR amplification (HiSxript II One-Step qRT-PCR SYBR Green kit; Vazyme). The following primer pairs targeting the SARS-CoV-2 S gene and SARSr-CoV nucleocapsid (N) gene were used (45 (link)): RBD-qF1 (5′-CAATGGTTTAACAGGCACAGG -3′)/RBD-qR1 (5′-CTCAAGTGTCTGTGGATCACG -3′) and NP-qF1 (5′-TCGTATGGGTCGCAACTGAG -3′)/NP-qR1 (5′-GCGAGAAGAGGCTTGACTGC -3′). PCRs were run according to the manufacturer’s instructions.
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