Mx 3000p real time pcr system

Total RNA was extracted by 1 ml TRIzol (Merck & Co., Inc., Kenilworth, NJ, USA) adapted to the manufacturer's protocol. 500 µg RNA was reverse transcribed to cDNA in 20 μl system by First Strand cDNA Synthesis Kit for RT-PCR (Merck & Co., Inc., Kenilworth, NJ, USA). Real-time PCR was performed using the Mx3000P real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). PCR was carried out as follows: 40 cycles of 94 °C for 15 s, 60 °C for 10 s, and 72 °C for 20 s. All procedures were performed independently three times. Gene expression was normalized to the GAPDH to calculate relative expression using the 2^−ΔΔCq method [18 (link)]. The primer sequences used in this study were listed as below: HAND2-AS1, forward: 5′-GGGTGTTTACGTAGACCAGAACC-3′; reverse: 5′-CTTCCAAAAGCCTTCTGCCTTAG-3′; GAPDH: forward:5′-GTCTCCTCTGACTTCAACAGCG-3′; reverse: 5′-ACCACCCTGTTGCTGTAGCCAA-3′; miR-146: forward: 5′-GAGAACTGAATTCCATGG-3′; reverse: 5′-GAACATGTCTGCGTATCTC-3′; U6: forward: 5′-CGAGCACAGAATCGCTTCA-3′; reverse: 5′-CTCGCTTCGGCAGCACATAT-3′; RARB, forward: 5′-GGTTTCACTGGCTTGACCATCG-3′; reverse: 5′-CCGTCTGAGAAAGTCATGGTGTC-3′; COX-2, forward: 5′-CGGTGAAACTCTGGCTAGACAG-3′; reverse: 5′-GCAAACCGTAGATGCTCAGGGA-3′; Caspase 3, forward: 5′-TGGCCCTGAAATACGAAGTC-3′; reverse: 5′-GGCAGTAGTCGACTCTGAAG-3′.

Total RNA was isolated with an RNA Isolation Kit (QIAGEN N.V. Hilden, German) according to the manufacturer’s protocol. One milligram RNA was reverse transcribed to cDNA by QuantiTect Reverse Transcription Kit (QIAGEN N.V. Hilden, German). Real-time PCR was carried out with the Mx3000P real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The protocols were as follows: 40 cycles of 94 °C for 15 s, 60 °C for 10 s, and 72 °C for 20 s. All procedures repeated thrice. Gene expression was normalized to the GAPDH to calculate relative expression using the 2^−ΔΔCq method^{17 (link)}. Primers for detecting lncARSR, MRP1, SURVIVIN, MMP2 AKT, and mTOR, listed below:
LncARSR, forward: 5ʹ-TTTGAAATGCTCTTTGAGGGAT-3ʹ; reverse: 5ʹ-TGCAGGTTGTCTGAAGTTGGA-3ʹ. MRP1, forward: 5ʹ-ACCCTAATCCCTGCCCAGAG-3ʹ, reverse: 5ʹ-CGCATTCCTTCTTCCAGTTC-3ʹ. SURVIVIN, forward: 5′-AAGAACTGGCCCTTCTTGGA-3ʹ, reverse: 5′-CAACCGGACGAATGCTTTT. MMP2, forward: 5ʹ-AGCGAGTGGATGCCGCCTTTAA-3ʹ, reverse: 5ʹ-CATTCCAGGCATCTGCGATGAG-3ʹ. AKT, forward: 5ʹ-TGGACTACCTGCACTCGGAGAA-3ʹ, reverse: 5ʹ-GTGCCGCAAAAGGTCTTCATGG-3ʹ. MTOR, forward: 5ʹ-AGCATCGGATGCTTAGGAGTGG-3ʹ, reverse: 5ʹ-CAGCCAGTCATCTTTGGAGACC-3ʹ. GAPDH, forward: 5ʹ-GTCTCCTCTGACTTCAACAGCG-3ʹ, reverse: 5ʹ-ACCACCCTGTTGCTGTAGCCAA-3ʹ.

The expression of HSPA5, HSPA1A, CHIP, TGF-β1, and Foxp3 mRNAs was quantified by qRT-PCR. The total RNA in colon tissues was extracted by Trizol (Invitrogen, USA). The cDNA was synthesized by reverse transcription kit (TaKaRa, Japan). The reaction was performed on the Mx3000P real-time PCR system (Thermo Fisher) for qRT-PCR using SYBR Green SuperMix (Roche, Basel, Switzerland) [6 ]. PCR conditions were shown below, 15 s under 94°C, 10 s under 60°C, and 20 s under 72°C for altogether 40 cycles. Subsequently, 2^−∆∆Ct approach was utilized for data analysis, with beta-actin as the internal reference. The primers used in this study are listed in Table 1.

For qRT-PCR analysis, total RNA was isolated by homogenization of lung tissue in RNAzol reagent (Fa. Biozym, Hessisch Oldendorf, Germany). Reverse transcription was performed using a RevertAid H Minus Strand cDNA Synthesis Kit (Thermo Scientific®, Waltham, MA, USA) according to the manufacturer's recommendations. qRT-PCR was performed on a TaqMan 9700HT system (Roche®, Indianapolis, IN, USA) and an Agilent Stratagene Mx3000P Real-Time PCR system (Santa Clara, CA, USA) using Maxima® SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Waltham, MA, USA). The sequences of the primers for the rat oligonucleotides were designed based on the NCBI GenBank. The qPCR primer sequences are presented in Additional File 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/6578578.

TRIzol (Invitrogen) was applied to collect total cellular and tissue RNAs, and later the collected RNAs were prepared into cDNA through reverse transcription using PrimeScript RT Kits (Invitrogen). This work utilized the Mx3000P real-time PCR system (Thermo Fisher) for qRT-PCR using SYBR Green SuperMix (Roche, Basel, Switzerland). PCR conditions were shown below, 15 s under 94°C, 10 s under 60°C, and 20 s under 72°C for altogether 40 cycles. Subsequently, 2^-∆∆Ct approach was utilized for data analysis, with GAPDH and U6 being the internal references. All primers are listed in Table 2.

Total RNA was extracted by 1 mL TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Then 1 mg RNA was reverse transcribed to cDNA in 20 μl system by the RT reaction kit (Promega), was performed using the Mx3000P real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). PCR was carried out as follows: 40 cycles of 94 °C for 15 s, 60 °C for 10 s, and 72 °C for 20 s. All procedures were repeated thrice. Gene expression was normalized to the GAPDH to calculate relative expression using the 2^−ΔΔCq method^{15 (link)}. The primer sequences used in this study were listed as below: circ_001621, divergent primers: forward: 5′-GCCAATATGAGCCAG-3′; reverse, 5′-CTTTCTTGGGAATCCAG-3′; GAPDH: divergent primers: forward: 5′-TCCCCCACCACACTGAATCT-3′; reverse, 5′-AACAGGAGGAGCAGAGAGCG-3′; miR-578: forward: 5′-GTGCAGGGTGTTAGGA-3′; reverse, 5′-GAAGAACACGTCTGGT-3′; U6: forward: 5′-CGAGCACAGAATCGCTTCA-3′; reverse, 5′-CTCGCTTCGGCAGCACATAT-3′; VEGF, forward: 5′-GGACCCGATGCGGTTAGAG-3′; reverse, 5′-ATCAAGTGGATGCCCCACAG-3′; CDK4, forward: 5′-GATGCGCCAGTTTCTAAGAGG-3′; reverse, 5′-GGTCGGCTTCAGAGTTTC-3′; MMP9, forward: 5′-CGCATCTGGGGCTTTAAACAT-3′; reverse, 5′-TCAGCACAAACAGGTTGCAG-3′; β-actin, forward: 5′-CACAGAGCCTCGCCTTTGCC-3′; reverse, 5′-ACCCATGCCCACCATCACG-3′.