Total RNA was isolated using RNAprep pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China), and reverse-transcribed into complementary DNA was performed using first complementary DNA Synthesization Kit with oligo(dT)15 (Tiangen Biotech). The levels of mRNA of genes of interest were measured by real-time PCR (7900 HT by Applied Biosystems, Foster City, CA, USA) using SYBR Green Master Mix (Roche Diagnostics, Indianapolis, IN, USA). Total amount of mRNA was normalized to endogenous β-actin mRNA. Sequences of PCR primer pairs were listed in Supplementary Table 1 .
Oligo dt 15
Manufactured by Tiangen Biotech
Sourced in China
Oligo(dT)15 is a synthetic oligonucleotide consisting of 15 deoxythymidine (dT) residues. It is commonly used as a primer for reverse transcription reactions in the process of cDNA synthesis from mRNA templates.
Automatically generated - may contain errors
3 protocols using oligo dt 15
1
Quantifying Gene Expression via RT-PCR
2
Characterization of Hepatic Progenitor Cells
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The cells cultured in suspension for >12 days were used in this procedure. Total RNA was extracted with TRIzol Reagent (Invitrogen) according to the instructions. Reverse transcription, in duplicate, was conducted by following the instructions of Oligo d(T)15 (TIANGEN, China) and TIANScript M-MLV kit (TIANGEN). The 2×Taq PCR Mix (Aidlab, China) was used in PCR amplification. Each PCR run included non-template control. The primers for fetal liver cell markers (CD34, CD90/Thy-1, CD117/c-Kit, CD133/prominin-1), early hepatic lineage (biliary) markers (AFP, CK19) and late hepatic lineage (hepatocyte) markers (ALB, CK18) were used 16 (link). The primers for the markers that associated with pluripotency including Oct4, Nanog, SMO, Bmi1, Notch1, β-catenin were used (the primers and cycling conditions refer Supplementary Table 1 ).
3
Quantitative PCR Analysis of Gene Expression
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Total RNA was isolated using RNAprep pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China), and first-strand cDNA synthesis was performed using the 1st cDNA Synthesis Kit with oligo(dT)15 (Tiangen Biotech). The mRNA levels were measured by real-time PCR using SYBR Green Master Mix (Roche Diagnostics, Indianapolis, IN, USA). The total amount of mRNA was normalized to endogenous β-actin mRNA.
Sequences of PCR primer pairs were as follows: TβRI, forward primer 5′-TGTTGGTACCCAAGGAAAGC-3′ and reverse primer 5′-CACTCTGTGGTTTGGAGCAA-3′; α-SMA, forward primer 5′-CAGGGAGTAATGGTTGGAAT-3′ and reverse primer 5′-TCTCAAACATAATCTGGGTCA-3′; inducible nitric oxide synthase (iNOS), forward primer 5′-CAGCTGGGCTCAACAAACCTT-3′ and reverse primer 5′-CATTGGAATGGAAGCGTATCG-3′; and β-actin, forward primer 5′-GCTATTTGGCGCTGGACTT-3′, and reverse primer 5′-GCGGCTCGTAGCTCTTCTC-3′.
Sequences of PCR primer pairs were as follows: TβRI, forward primer 5′-TGTTGGTACCCAAGGAAAGC-3′ and reverse primer 5′-CACTCTGTGGTTTGGAGCAA-3′; α-SMA, forward primer 5′-CAGGGAGTAATGGTTGGAAT-3′ and reverse primer 5′-TCTCAAACATAATCTGGGTCA-3′; inducible nitric oxide synthase (iNOS), forward primer 5′-CAGCTGGGCTCAACAAACCTT-3′ and reverse primer 5′-CATTGGAATGGAAGCGTATCG-3′; and β-actin, forward primer 5′-GCTATTTGGCGCTGGACTT-3′, and reverse primer 5′-GCGGCTCGTAGCTCTTCTC-3′.
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