Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified by NanoDrop 2000C spectrophotometer (Thermo, Wilmington, DE, USA). In brief, total RNA and miRNA were synthesized into cDNA using PrimeScript RT reagent kit with gDNA Eraser and Mir-X miRNA First-Strand Synthsis Kit (TaKaRa, Kusatsu, Shiga, Japan). Quantitative real-time PCR (qRT-PCR) was then applied using SYBR Premix Ex Taq II (TaKaRa, Kusatsu, Shiga, Japan) in the ABI 7500 qRT-PCR System with the following primers: α-ENaC forward (5′-AAC AAA TCG GACTGC TTC TAC-3′) and reverse (5′-AGC CAC CAT CAT CCA TAA A-3′), β-ENaC forward (5′-GGG ACC AAA GCA CCA AT-3′) and reverse (5′-CAG ACG CAG GGA GTC ATAG-3′), γ-ENaC forward (5′-GCACCG TTC GCC ACC TTC TA-3′) and reverse (5′-AGG TCA CCA GCA GCT CCT CA-3′), and GAPDH forward (5′-AGA AGG CTG GGG CTC ATT TG-3′) and reverse (5′-AGG GGC CAT CCA CAG TCT TC-3′). Relative expression of mRNA/miRNA was calculated using the 2−Δ(ΔCT) method, and GAPDH/U6 was used as a reference.
Primescript rt reagent kit with gdna eraser and mir x mirna first strand synthsis kit
Manufactured by Takara Bio
Sourced in Japan
The PrimeScript RT reagent kit with gDNA Eraser and Mir-X miRNA First-Strand Synthesis Kit is a laboratory equipment product designed for reverse transcription and miRNA first-strand synthesis. The kit includes reagents for genomic DNA removal and cDNA synthesis from both mRNA and miRNA.
Automatically generated - may contain errors
2 protocols using primescript rt reagent kit with gdna eraser and mir x mirna first strand synthsis kit
1
qRT-PCR Analysis of ENaC Subunits
2
Quantification of ENaC Expression
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Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified by NanoDrop 2000C spectrophotometer (Thermo, Wilmington, DE, USA). In brief, total RNA and miRNA were synthesized into cDNA using PrimeScript RT reagent kit with gDNA Eraser and Mir-X miRNA First-Strand Synthsis Kit (TaKaRa, Kusatsu, Shiga, Japan). Quantitative real-time PCR (qRT-PCR) was then applied using SYBR Premix Ex Taq Ⅱ (TaKaRa, Kusatsu, Shiga, Japan) in the ABI 7500 qRT-PCR System with the following primers: α-ENaC forward (5′-AAC AAA TCG GACTGC TTC TAC-3′) and reverse (5′-AGC CAC CAT CAT CCA TAA A-3′), β-ENaC forward (5'-GGG ACC AAA GCA CCA AT-3') and reverse (5'-CAG ACG CAG GGA GTC ATAG-3'), γ-ENaC forward (5′-GCACCG TTC GCC ACC TTC TA-3′) and reverse (5′-AGG TCA CCA GCA GCT CCT CA-3′), and GAPDH forward (5′-AGA AGG CTG GGG CTC ATT TG-3′) and reverse (5′-AGG GGC CAT CCA CAG TCT TC-3′). Relative expression of mRNA/miRNA was calculated using the 2 -Δ(ΔCT) method, and GAPDH/U6 was used as a reference.
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