Total RNA was isolated from cell culture lysates using RNeasy mini kit (Qiagen, Cat# 74104), in accordance with the manufacturer’s instructions. Total nucleic acid concentrations were quantified using the Nanodrop 2000 (Thermo Fisher Scientific). RT-PCR was performed with a total of 100 ng of RNA using the qScript XLT 1-Step RT-qPCR ToughMix Low ROX (Quantabio, Cat# 89236-676) reaction mix and the Applied Biosystems 7500 system (Applied Biosystems, Foster City, CA, USA). TaqMan Gene Expression Assays and the following primers were used: Hs00208382_m1: Mfn2 primer; Hs00250475_m1: Mfn1 primer; Hs00953477_m1: SIRT3 primer; Hs00697394_g1: Mff primer; Hs01047018_m1: OPA1 primer; Hs00608023_m1: Bcl-2 primer; and DNM1L-Hs01552605_m1: Drp-1 primer. Human GAPDH (ThermoFisher Scientific, Cat# 4310884E) was used as an endogenous control for sample normalization. PCR product specificity was determined using melting curve assessment and gene expression differences were determined using the ΔΔCt method.
Qscript xlt 1 step rt qpcr toughmix low rox
Manufactured by Quantabio
Sourced in United States
The QScript XLT 1-Step RT-qPCR ToughMix Low ROX is a ready-to-use solution for one-step reverse transcription and real-time quantitative PCR (RT-qPCR) analysis. It contains all the necessary components for efficient RNA detection and quantification.
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Lab products found in correlation
4 protocols using qscript xlt 1 step rt qpcr toughmix low rox
1
Mitochondrial Dynamics Gene Expression
2
Quantitative RNA Expression Analysis
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Total RNA was isolated using NucleoSpin RNA XS (Takara Cat #740902.50) following the manufacturer’s instructions and quantified using the Nanodrop 2000 (Thermo Fisher Scientific). RT-PCR was performed with a total of 100 to 800 ng of RNA using the qScript XLT 1-Step RT-qPCR ToughMix Low ROX (Quantabio, Cat #89236–676) reaction mix and the Applied Biosystems 7500 system (Applied Biosystems, Foster City, CA). TaqMan Gene Expression Assays and the following primers were used for gene amplification: Hs00154952_m1 (EIF4G2), Hs00793604_m1 (YWHAB), Hs00947931_m1 (VAC14), Hs01076090_m1 (EGFR), Hs01635324_s1 (HMGN2), Hs01027550_g1 (AKIP1), Hs00197576_m1 (DNPH1), Hs00169098_m1 (APP), and Hs00705137_s1 (IFITM1). HIV-1 gag was measured using the following primers and probe: HIV-1gag_F 5′-GACATAAGACAGGGACCAAAGG-3′; HIV-1gag_R 5′-CTGGGTTTGCATTTTGGACC-3′; HIV-1gag_Probe 5′-AACTCTAAGAGCCGAGCAAGCTTCAC-3′. Human GAPDH was calculated for sample normalization.
3
SARS-CoV-2 Host Factor Expression Analysis
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Total RNA was isolated from cell culture lysates using RNeasy mini kit (Qiagen, Germantown, MD, Cat # 74104) following the manufacturer’s instructions and quantified using the Nanodrop 2000 (Thermo Fisher Scientific). RT-PCR was performed with a total of 100–800 ng of RNA using the qScript XLT 1-Step RT-qPCR ToughMix Low ROX (Quantabio, Beverly, MA, USA, Cat #89236–676) reaction mix and the Applied Biosystems 7500 system (Applied Biosystems, Foster City, CA). TaqMan Gene Expression Assays and ACE2 primer: Hs01085333_m1; TMPRSS2 primer: Hs00237175_m1; ADAM17 primer: Hs01041915_m1; BSG primer: Hs00936295_m1; DPP4 primer: Hs00897386_m1; AGTR2 primer: Hs02621316_s1; ANPEP primer: Hs00174265_m1; CATHEPSIN L primer: Hs02803063_cn and CATHEPSIN B primer: Hs02148115_cn were used for gene amplification. HIV-1 gag was measured using the following primers and probe: HIV-1gag_F 5′-GACATAAGACAGGGACCAAAGG-3′; HIV-1gag_R 5′-CTGGGTTTGCATTTTGGACC-3′; HIV-1gag_Probe 5′-AACTCTAAGAGCCGAGCAAGCTTCAC-3′. Human GAPDH was calculated for sample normalization. Coveted PCR product specificity was determined using melting curve assessment and gene expression fluctuations were determined by the ΔΔCt method, with Ct as the cycle number at threshold.
4
Quantifying SARS-CoV-2 Host Factors by RT-qPCR
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Total RNA was isolated from cell culture lysates using RNeasy mini kit (Qiagen, Germantown, MD, Cat # 74104) following the manufacturer’s instructions and quantified using the Nanodrop 2000 (Thermo Fisher Scientific). RT-PCR was performed with a total of 100-800 ng of RNA using the qScript XLT 1-Step RT-qPCR ToughMix Low ROX (Quantabio, Beverly, MA, USA, Cat #89236-676) reaction mix and the Applied Biosystems 7500 system (Applied Biosystems, Foster City, CA). TaqMan Gene Expression Assays and ACE2 primer: Hs01085333_m1; TMPRSS2 primer: Hs00237175_m1; ADAM17 primer: Hs01041915_m1; BSG primer: Hs00936295_m1; DPP4 primer: Hs00897386_m1; AGTR2 primer: Hs02621316_s1; ANPEP primer: Hs00174265_m1; CATHEPSIN L primer: Hs02803063_cn and CATHEPSIN B primer: Hs02148115_cn were used for gene amplification. HIV-1 gag was measured using the following primers and probe: HIV-1gag_F 5′-GACATAAGACAGGGACCAAAGG-3′; HIV-1gag_R 5′-CTGGGTTTGCATTTTGGACC-3′; HIV-1gag_Probe 5′-AACTCTAAGAGCCGAGCAAGCTTCAC-3′. Human GAPDH was calculated for sample normalization. Coveted PCR product specificity was determined using melting curve assessment and gene expression fluctuations were determined by the ΔΔCt method, with Ct as the cycle number at threshold.
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