Total RNA from BEAS-2B cells was extracted by TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (2 µg) was then reverse-transcribed into cDNA with the Transcriptor 1st strand cDNA Synthesis Kit (Roche Diagnostics). Real-time qPCR was performed using the OneStep TB Green RT-PCR Kit (Takara Bio, Inc.). The primers used are listed in Table I . The thermocycling program was 95˚C for 10 min followed by 40 cycles of 95˚C for 15 sec and 60˚C for 60 sec. The relative expression of each gene was calculated by the 2-ΔΔCq method (13 (link)) and GAPDH was used as the internal control.
Onestep tb green rt pcr kit
Manufactured by Takara Bio
Sourced in Japan
The OneStep TB Green RT-PCR Kit is a real-time RT-PCR (reverse transcription polymerase chain reaction) kit designed for the detection of Mycobacterium tuberculosis in clinical samples. The kit includes all the necessary reagents for a one-step RT-PCR reaction, including a reverse transcriptase enzyme and TB Green dye for fluorescent detection.
Automatically generated - may contain errors
3 protocols using onestep tb green rt pcr kit
1
RNA Extraction and qPCR Analysis Protocol
2
SARS-CoV-2 Viral Load Quantification
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A viral load reduction assay was performed on VeroE6-TMPRSS2 cells by RT‒qPCR as described previously.42 (link) The cell culture supernatants or animal tissue homogenates were lysed in RLT buffer, followed by RNA extraction using a RNeasy mini kit (Qiagen). The viral load was quantified by a one-step TB green RT‒PCR kit (Takara) according to the protocol of the LightCycle 480 real-time PCR system (Roche). The primers used for quantification targeted SARS-CoV-2 RdRp genes: forward sequence: 5′-CGCATACAGTCTTRCAGGCT-3’; reverse sequence: 5′-GTGTGATGTTGAWATGACATGGTC-3’.
3
RNA Isolation and qRT-PCR Analysis
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Lung tissue or cells were lysed by TRIzol kit (QIAGEN) and RNA was isolated per the manufacturer’s instructions. In addition, the PCR was carried out by the One Step TB Green RT-PCR Kit (TaKaRa, Japan) as previously described. The relative expression of each gene was calculated using the 2−ΔΔCT method after correction by GAPDH expression. All primer sequences are listed in the Supplemental Table 1 .
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