For determination of mRNA expression levels, total RNA was recovered using TriFast Reagent (Peqlab, Erlangen, Germany) according to the manufacturer's recommendations. The primers (Metabion, Martinsried, Germany) used in these experiments were: Aldose reductase (AR)_fw: 5′-ATC GCA GCC AAG CAC AAT AA-3′; AR_rev: 5′-AGC AAT GCG TTC TGG TGT CA-3′; TonEBP/NFAT5_fw: 5′-AAT CGC CCA AGT CCC TCT AC-3′; TonEBP/NFAT5_rev: 5′-GGT GGT AAA GGA GCT GCA AG -3′; actin_fw: 5′- CCA ACC GCG AGA AGA TGA-3′; actin_rev: 5′- CCA GAG GCG TAC AGG GAT AG -3′. Experiments were performed on a Roche LightCycler 480, using the SensiMix SYBR One-Step Kit (Bioline, Luckenwalde, Germany) according to the manufacturer's recommendations. Relative mRNA expression of the respective genes was calculated by the 2−ΔΔCT–method (Livak and Schmittgen, 2001 (link)), using β-actin as housekeeping gene. Specificity of PCR productformation was confirmed by monitoring melting point analysisand by agarose gel electrophoresis as described (Küper et al., 2012b (link)).
Sensimix sybr one step kit
Manufactured by Meridian Bioscience
Sourced in Germany
The SensiMix SYBR One-Step Kit is a reagent designed for reverse transcription and real-time quantitative PCR (RT-qPCR) analysis. It contains all the necessary components to perform one-step RT-qPCR in a single reaction mix, including reverse transcriptase, hot-start DNA polymerase, and SYBR Green I dye for detection.
Automatically generated - may contain errors
5 protocols using sensimix sybr one step kit
1
Quantification of mRNA Expression Levels
2
Quantitative Analysis of mRNA Expression
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For determination of mRNA expression levels, total RNA from IMCD-3 cells was recovered using TriFast Reagent (Peqlab, Erlangen, Germany) according to the manufacturer's recommendations. The primers (Metabion, Martinsried, Germany) used in these experiments were:
HSP70_fw: 5′-tga gtc cca cac tct cac ca-3′;
HSP70_rev: 5′-ctg tgg gtg aag ctg tta agg -3′;
NFAT5_fw: 5′-AAT CGC CCA AGT CCC TCT AC-3′;
NFAT5_rev: 5′-GGT GGT AAA GGA GCT GCA AG -3′;
actin_fw: 5′- CCA ACC GCG AGA AGA TGA-3′;
actin_rev: 5′- CCA GAG GCG TAC AGG GAT AG -3′.
3
Gene Expression Analysis in Kidney Cell Lines
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For determination of NFAT5, S100A4, AR, and β-Actin mRNA expression levels, the total RNA from HK-2 or CaKi-1 cells was prepared by adding TRIFAST Reagent (Peqlab, Erlangen, Germany). The primers (Metabion, Martinsried, Germany) used in this experiment are:
NFAT5_fw: 5′- AAT CGC CCA AGT CCC TCT AC -3′;
NFAT5_rev: 5′- GGT GGT AAA GGA GCT GCA AG -3′;
Actin_fw: 5′- CCA ACC GCG AGA AGA TGA -3′;
Actin_rev: 5′- CCA GAG GCG TAC AGG GAT AG -3′;
S100A4_fw: 5′-CGC TTC TTC TTT CTT GGT TTG-3′;
S100A4_rev: 5′-GAG TAC TTG TGG AAG GTG GAC A-3′;
AR_fw: 5′ATC CGA GCC AAG CAC AAT AA -3′;
AR_rev: 5′-AGC AAT GCG TTC TGG TGT CA -3′
Experiments were caried out on a Roche LightCycler 480 using the SensiMix SYBR One-Step Kit (Bioline, Luckenwalde, Germany) according to the manufacturer's recommendations. Specificity of PCR product formation was confirmed by melting point analysis and by agarose gel electrophoresis.
NFAT5_fw: 5′- AAT CGC CCA AGT CCC TCT AC -3′;
NFAT5_rev: 5′- GGT GGT AAA GGA GCT GCA AG -3′;
Actin_fw: 5′- CCA ACC GCG AGA AGA TGA -3′;
Actin_rev: 5′- CCA GAG GCG TAC AGG GAT AG -3′;
S100A4_fw: 5′-CGC TTC TTC TTT CTT GGT TTG-3′;
S100A4_rev: 5′-GAG TAC TTG TGG AAG GTG GAC A-3′;
AR_fw: 5′ATC CGA GCC AAG CAC AAT AA -3′;
AR_rev: 5′-AGC AAT GCG TTC TGG TGT CA -3′
Experiments were caried out on a Roche LightCycler 480 using the SensiMix SYBR One-Step Kit (Bioline, Luckenwalde, Germany) according to the manufacturer's recommendations. Specificity of PCR product formation was confirmed by melting point analysis and by agarose gel electrophoresis.
4
Quantitative mRNA Expression Analysis
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For determination of NFAT5, AR, HSP70, BGT-1, and β-actin mRNA expression levels, tissues were homogenized in TRIFAST Reagent (Peqlab, Erlangen, Germany), and RNA was isolated as recommended by the manufacturer. Oligonucleotides used in this experiment are listed in Table 1 . Experiments were carried out on a Roche LightCycler 480, using the SensiMix SYBR One-Step Kit (Bioline, Luckenwalde, Germany) according to the manufacturer's recommendations. Specificity of PCR product formation was confirmed by monitoring melting point analysis and by agarose gel electrophoresis.
5
Quantification of SARS-CoV-2 Genomic and Subgenomic RNA
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Duplicate flasks were prepared so that RNA extraction and DMV analysis could be performed on samples from the same experiment. Total cellular RNA was extracted from infected cells 10 h after inoculation using an RNeasy minikit (Qiagen). The RNA integrity was analyzed with the 2100 Bioanalyzer using the RNA 6000 Nano kit (Agilent). Both cDNA synthesis and PCR were performed in a single tube from total RNA using the SensiMix SYBR one-step kit (Bioline). Genomic RNA was quantified by amplifying a 124-bp region from the nsp3 gene with the forward primer 5′ AATAAGCAGGAGGCAAATGTTC 3′ and the reverse primer 5′ CCCACAACACAATGAAACAAATC 3′. A 110-nt amplicon spanning the leader-body junction of sgRNA7 was amplified using forward primer 5′ GTACCCTCTCAACTCTAAAAC 3′ and reverse primer 5′ GCGGTTTACAGAGGAGCTT 3′. Amplifications were carried out in triplicate, with a 250 nM primer concentration in 25-µl reaction volumes. Melt curve analysis was performed to confirm PCR product specificity. Absolute quantitation was performed by comparison to a standard curve of cloned amplicons.
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