Total RNA was extracted from tissue samples isolated from patients with lung cancer or pulmonary bullae using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Total RNA (2 µg) was reverse transcribed (20 µl reaction volume) into cDNA using a reverse transcription system, as previously described (13 (link)). The following primers, designed using the Primer Premier software version 5.0 (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) were employed: CCBE1 forward, 5′-CGACTAAATACCCGTGTCTGAAG-3′ and reverse, 5′-TCGGCACAAACGTCGTAATCT-3′; β-actin forward, 5′-GCTCGTCGTCGACAACGGCTC-3′ and reverse, 5′-CAAACATGATCTGGGTCACTTCTC-3′. Amplification was performed under the following conditions: Initial incubation at 95°C for 10 sec, followed by 40 cycles at 95°C for 5 sec and at 62°C for 45 sec, and extension at 72°C for 3 min. The 25-µ LlPCR reaction system included 1 µl temple, 2 µl primers, 2 µl dNTP, 0.5 µl RT/Platinum™ Taq Mix (Invitrogen; Thermo Fisher Scientific, Inc.) and distilled water. PCR products were detected by 2% agarose gel electrophoresis as previously described (13 (link)).
Rt platinum taq mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RT/Platinum™ Taq Mix is a ready-to-use solution for reverse transcription and PCR amplification. It contains Platinum™ Taq DNA Polymerase and all the necessary components for cDNA synthesis and subsequent PCR, including dNTPs, MgCl2, and reaction buffer.
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2 protocols using rt platinum taq mix
1
Quantitative Gene Expression Analysis of CCBE1 in Lung Diseases
2
SARS-CoV-2 RNA Quantification by RT-qPCR
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The RNA of SARS-CoV-2 was quantified by RT–qPCR amplification of the nucleocapsid (N) and envelope (E) genes from the viral genome. The E gene was targeted by the E_Sarbeco primers and E_Sarbeco_P1 probe (Corman et al., 2020 (link)), and the N gene was targeted by the SARS-CoV-2 N1+N2 Assay Kit (Qiagen, Germany), which includes N1 and N2 primers and probes from the CDC design (Lu et al., 2020 (link)). The total volume of the RT–qPCR was 20 μL, and all measurements were performed in duplicate for each sample. The reaction mixture contained 5 μL of RNA, 10 μL of 2 × Reaction Mix from the SuperScript™ III One-Step RT–PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, USA), 16 nmol of additional MgSO4, 0.5 μL of SuperScript™ III RT/Platinum™ Taq Mix, and either of the two primer and probe sets. E_Sarbeco primers and probes were used at final concentrations of 0.4 nM and 0.2 nM, respectively. The N1+N2 Assay Kit was used at a 20× dilution. The thermal cycling conditions for both E gene and N gene reactions were as described by Corman et al. (2020) (link). Serial dilutions of the synthetic RNA SARS-CoV-2 Positive Run Control (Exact Diagnostics, USA) were used to produce a standard curve.
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