Iqtm5 multicolour real time pcr detection system

QRT-PCR reactions were carried out using a qSYBR-green-containing PCR kit (Qiagen, Germantown, MD, USA). The relative change was determined using the method of 2^-△△Ct. The U6 small nuclear RNA gene and GAPDH served as the internal controls for the qRT-PCR assays. The Bio-Rad (Hercules, CA, USA) IQTM5 Multicolour Real-Time PCR Detection System (USA) was used to perform RT-PCR assays. Primers used in this study were synthesized from Nanjing Genscript Inc. (Nanjing, China) and the sequences are listed in Table 2.

Total cellular RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Reverse transcription and qRT-PCR reactions were performed by means of a qSYBR-green-containing PCR kit (Qiagen, USA). The primers for LDHA were synthesized by Invitrogen: forward, 5′-TTGGTCCAGCGTAACGTGAAC-3′ and reverse, 5′-CCAGGATGTGTAGCCTTTGAG-3′. The threshold cycle (CT) value for LDHA was normalized against the CT value for control β-actin, while U6 snRNA was used as an internal control for the relative amount of miR-34a. The relative fold-change in expression with respect to a control sample was calculated by the 2^−ΔΔCt method. All of the real-time PCR assays were performed with the Bio-Rad IQTM5 Multicolour Real-Time PCR Detection System (USA).

Total RNA was extracted from the cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). A NanoDrop ND-1000 instrument was used to determine the concentrations of the RNA samples. The integrity of RNA was assessed by electrophoresis on a denaturing agarose gel. Reverse transcription and qRT-PCR reactions were performed using a QuantiFast SYBR® Green RT-PCR Kit (QIAGEN, Germantown, MD, USA), which is a one-step RT-PCR kit. Each reaction was performed in triplicate. The primer sequences are given as follows: AHNAK: 5′-ATGCTCCAGGGCTCAACCT-3' (forward) and 5'-CGTGCCCCAACGTTAAGCTT-3' (reverse); β-actin: 5'-CGCGAGAAGATGACCCAGAT-3' (forward) and 5′-GGGCATACCCCTCGTAGATG-3' (reverse); Wnt1: 5'-ATGGGGCTCTGGGCGCTGTTG-3' (forward) and 5'-TCACAGACACTCGTGCAGTAC-3' (reverse); c-Myc: 5’-AGAAATGTCCTGAGCAATCACC-3' (forward) and 5’-AAGGTTGTGAGGTTGCATTTGA-3' (reverse); β-catenin: 5′- CCGCATGGAAGAAATAGTTGAAG-3′ (forward) and 5′- CAATTCGGTTGTGAACATCCC-3′ (reverse). The real-time PCR assays were performed with the Bio-Rad IQTM5 Multicolour Real-Time PCR Detection System (USA). The specificity of the amplification products was confirmed by melting curve analysis. The values were normalized to internal controls and fold changes were calculated through relative quantification (2^-ΔΔCt).

Total RNA was isolated using TRIzol reagent (Life Technologies, USA). The nuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Complementary DNA was synthesized using the PrimeScript RT reagent kit (Takara Bio Inc., China), and RT-PCR was performed using SYBR Premix Ex Taq (Takara Bio Inc.). The primers for circGFRA1 are F: 5′-CCTCCGGGTTAAGAACAAGC-3′, R: 5′-CTGGCTGGCAGTTGGTAAAA-3′. The primers for GFRA1 are F: 5′-CCAAAGGGAACAACTGCCTG-3′, R: 5′-CGGTTGCAGACATCGTTGGA-3′. The threshold cycle (CT) value for circGFRA1 or GFRA1 was normalized against the CT value for control β-actin, while U6 snRNA was used as an internal control for the relative amount of miR-34a. The relative fold-change in expression with respect to a control sample was calculated by the 2-ΔΔCt method. All the real-time PCR assays were performed with the Bio-Rad IQTM5 Multicolour Real-Time PCR Detection System (USA).

The total RNA from cells or tissues was extracted with TRIzol reagent (Invitrogen, USA). Reverse transcription and qRT-PCR reactions were performed using a qSYBR-green-containing PCR kit (Qiagen, USA). The threshold cycle (CT) value for MTDH and GABARAPL1 were normalized against the CT value for internal control β-actin. The fold change was determined as 2-ΔΔCt. The primers for qRT-PCR detection were synthesised by Invitrogen: MTDH forward, 5’-TGGCAAATGTGGCCAACA-3’; reverse, 5’-TATTAGGTAACCGACCCCCTCTT-3’. GABARAPL1 forward, 5’-CCCTCCCTTGGTTATCATCCA-3’; reverse, 5’-ACTCCCACCCCACAAAATCC-3’. All the qRT-PCR assays were performed with the Bio-Rad IQTM5 Multicolour Real-Time PCR Detection System (USA).

The total RNA from all cell lines and tissues were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, United States). Reverse transcription and qRT-PCR reactions were performed with qSYBR-green-containing PCR kit (Qiagen, United States). The threshold cycle value (CT, the fractional cycle number at which the fluorescence of each sample passes the fixed threshold) of SOX2 was normalized against the CT value of internal control β-actin. The fold change was determined as 2^-ΔΔCt. The primers for qRT-PCR detection were synthesized by Invitrogen. SOX2 forward, 5′-TAATTAGAATTCATGTA CAACATGATGGAGACG-3′, reverse, 5′-TAATTAGGTACCT CACATGTGTGAGAGGGGCAGTGTGC-3′. The detection was performed with Bio-Rad IQTM5 Multicolour Real-Time PCR Detection System (United States).