qScript cDNA Synthesis Kit

Protocol tips

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	When required, cDNA product can be diluted with 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA

Protocol tips
When required, cDNA product can be diluted with 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA

Publication protocol

Total RNA was isolated from liver tissues or cells using TRI Reagent (Sigma-Aldrich), according to the manufacturer’s instruction. cDNA was generated by Reverse transcription, using 1 μg RNA and qScript cDNA Synthesis kit (Quanta, Bioscience Inc) in 20 μL. Samples were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), using Perfecta mix reaction ROX Form Quanta (Bioscience Inc.), according to the manufacturer’s instructions. All data are normalized to Hprt1 expression and expressed as Relative Quantification (method of 2^−ΔΔCt) or as −ΔCt (i.e., Ct Hprt − Ct target, the higher is the −ΔCt, the greater is the amount of target amplicon). Primers used for human cells were: Hs Hamp1 forward, 5’-CCA-GCT-GGA- TGC-CCA-TGT-T-3’, and reverse, 5’-GCC-GCA-GCA-GAA-AATGCA- 3’; Hs Hprt1 forward, 5’-TGC-TTT-CCT-TGG-TCA-GGC-AG-3’, and reverse, 5’-AAG-CTT-GCG-ACC-TTG-ACC-AT-3’; Hs Id1 forward, 5’-GTA-AAC-GTG-CTG-CTC-TAC-GAC-ATG-A-3’, and reverse, 5’-AGCTCC-AAC-TGA-AGG-TCC-CTG-A-3’; Hs Soc3 forward 5’-CAG-GAA-TGT-AGC-AGC-GAT-GGA-A-3′ and reverse 5′-CCT-GTC-CAG-CCC-AAT-ACC-TGA-3′; Hs HPA1 forward, 5’-TAC-CTT-CAT-TGC-ACA-AAC-ACT-G-3’ and reverse, 5’-ACT-TGG-TGA-CAT-TAT-GGA-GGT-T-3’; Hs Fpn forward 5’ ATC-CAT-GTG-CGT-GGA-GTA-CG-3’ and reverse 5’- AGG-GGT-TTT-GGC-TCA-GTA-TCT-TT-3’; Hs TfR1 forward 5’-TTT-CCA-CCA-TCT-CGG-TCA-TC-3’and reverse 5’-GGG-ACA-GTC-TCC-TTC-CAT-ATT-C-3’; Hs Zip14 forward 5’-CCT-GCT-TGG-CTT-ATG-GAG-AA-3’and reverse 5’-CCT-CGC-CAT-ACC-GAT-GTA-TTA-G-3’; Hs Bmp6 forward 5’-GGT-CTC-CAG-TGC-TTC-AGA-TTA-C-3’ and reverse 5’-CAG-GTC-TTG-GAA-ACT-CAC-ATA-CA-3’.

The primers for qPCR assay for mouse liver tissues were: Mm Hamp1 forward, 5’-AAG-CAG-GGC-AGA-CAT-TGC-GAT-3’, and reverse, 5’-CAG-GAT-GTG-GCT-CTA-GGC-TAT-GT-3’; Mm Hprt1 forward, 5’-CTG-GTT-AAG-CAG-TAC-AGC-CCC-AA-3’, and reverse, 5’-CAGGAG-GTC-CTT-TTC-ACC-AGC-3’; Mm Id1 forward, 5’-ACC-TG-AACGGC-GAG-ATC-A-3’, and reverse, 5’-TCG-TCG-GCT-GGA-ACA-CAT-G-3’; Mm Bmp6 forward, 5’-GAA-CCT-GGT-GGA-GTA-CGA-CAA-3’, and reverse, 5’-TCC-TTG-TAG-ACG-CGG-AAC-TC-3’; Mm TfR1 forward, 5’- AGC-CAG-ATC-AGC-ATT-CTC-TAA-C3’, and reverse, 5’-TCT-GCA-GCC-AGT-TTC-ATC-TC-3’; Mm Fpn forward, 5’- CAT-TGG-TGA-CTG-GGT-GGA-TAA-G-3’, and reverse, 5’-CAG-GAG-CTC-ATT-CTT-GTG-TAG-G-3’; Mm Zip14 forward, 5’- CAT-GGA-CCG-CTA-TGG-AAA-GAA-3’, and reverse, 5’- CCT-TGG-GCT-GGG-AAA-CAT-TA-3’; Mm Socs3 forward, 5’-TTA-AAT-GCC-CTC-TGT-CCC-AGG-3’, and reverse, 5’ TGT-TTG-GCT-CCT-TGT-GTG-CC-3’; Mm Crp forward, 5’-GCT-ACT-CTG-GTG-CCT-TCT-GAT-CA-3’, and reverse, 5’-GGC-TTC-TTT-GAC-TCT-GCT-TCC-A-3’.

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