Quantitect sybr green pcr system

Total RNA was isolated by using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA samples (1 μg each) were then reverse-transcribed into cDNA using the Omniscript reverse transcription kit (Qiagen, Germany). Quantitative real-time PCR reaction was performed by using the Quantitect SyBr green PCR system (Qiagen). RNAU6B snRNA (U6) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as miRNA and mRNA internal control, respectively. The specific forward primer of miR-27a-3p was 5ʹ-ATGGTTCGTGGGTTCACAGTGGCTAAGTTCCG-3ʹ. The specific forward primer of U6 was 5ʹ-ACGCAAATTCGTGAAGCGTT-3ʹ. Reverse primers for miR-27a-3p and U6 were provided by Qiagen. The primers for GATA6 and GAPDH were as follows: GATA6: 5ʹ-TTCCTACGCTTCGCATCC-3ʹ (sense), 5ʹ-TGGTCGAGGTCAGTGAACAGC-3ʹ (antisense); GAPDH: 5ʹ-GAGTCAACGGATTTGGTCGT-3ʹ (sense), 5ʹ-TTGATTTTGGAGGGATCTCG-3ʹ (antisense). The relative expression of GATA6 mRNA and miR-27a-3p was calculated by using 2^−ΔΔCt method.

Total RNA was extracted using Trizol (Invitrogen), according to the manufacturer's instructions. For mRNA analysis, cDNA was randomly primed from 2.0 μg total RNA using the Omniscript reverse transcription kit (Qiagen, Hilden, Germany). Real-time PCR was subsequently carried out in triplicate with a 1:4 dilution of cDNA using the Quantitect SyBr green PCR system (Qiagen) on a Rotorgene 6000 series PCR machine (Qiagen). Data were collected and analyzed using the Rotorgene software accompanying the PCR machine. Relative expression levels were determined using the comparative quantification feature of the Rotorgene software. All mRNA quantification data were normalized to GAPDH. For miRNA analysis, quantitative PCR was carried out as above, using TaqMan miRNA assays according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). Sequences of PCR primers and RNA oligonucleotides for real-time PCR were generated by Shanghai Sangon Company as previously described.(17 (link))

Total RNA was extracted from chick myotubes using TRIzol reagent (Invitrogen, USA) according to the manufacturer's directions. Complementary DNA was synthesized from total RNA using a random primer (TaKaRa, Shiga, Japan) and ReverTra Ace (TOYOBO, Tokyo, Japan). The sequences of the primers were as follows: atrogin-1/MAFbx, forward: 5′-CCAACAACCCAGAGACCTGT-3′ and reverse: 5′-GGAGCTTCACACGAACATGA-3′; microtubule-associated protein 1 light chain 3B (LC3B), forward: 5′-TCCGAGATCAGCATCCAACT-3′ and reverse: 5′-CACCATGCTGTGTCCGTTC-3′; GABA(A) receptor-associated protein like 1 (GABARAPL1), forward: 5′-CCGACAGAGTCCCCGTAATT-3′ and reverse: 5′-ATGGTAGCACTTGTGGGAGG-3′; autophagy-related 12 (ATG12), forward: 5′-CGGAAAGGACCCCAGAGAG-3′ and reverse: 5′-CTTGATGAAGTCGCACAGGC-3′; cathepsin B, forward: 5′-CAAGCTCAACACCACTGGAA-3′ and reverse: 5′-TCAAAGGTATCCGGCAAATC-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward: 5′-CCTCTCTGGCAAAGTCCAAG-3′ and reverse: 5′-CATCTGCCCATTTGATGTTG-3′; 18S rRNA, forward: 5′-AAACGGCTACCACATCCAAG-3′ and reverse: 5′-CCTCCAATGGATCCTCGTTA-3′. The mRNA levels were measured by real-time PCR analysis using a LightCycler^® instrument (Roche Diagnostics, Basel, Switzerland) and the QuantiTect SYBR Green PCR system (Qiagen, Hilden, Germany). The level of GAPDH or 18S rRNA was measured as an internal control.