Total RNA was extracted with/ isolated from cultured cells/ samples/ tumor tissues using/ by Trizol reagent (Invitrogen, Thermo Fisher Scientific, Braunschweig, Germany)/ RNeasy kit (Qiagen)/ RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol /following the manufacturer’s (recommended) instructions as described previously.
TRIzol reagent (Thermo Fisher Scientific) was used to extract RNA from samples.
第二句（可与第一句合并）
RNA (2 μg) was converted into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo).
Total RNA was reverse-transcribed into cDNA using the aScript cDNA SuperMix (Quanta Biosciences).
2 μg RNA was used to synthesize cDNA using the PrimeScript RTreagent kit (Takara, DRR037A).
第三句
Gene primers (…) were designed using Primer Express v3.0 Software.
The primer sequences used are listed/shown in Supplemental Experimental Procedures/ Table X. 也可写与句末。
The following primers were used:/ Gene-specific primers as follows: Gene名称forward 5′-XXX-3′ and reverse 5′- XXX-3′;
第四句
RT-PCR was performed with/ accomplished using/ carried out using …according to the manufacturer’s instructions. 第五句
GADPH /U6 was used as endogenous controls/ an internal reference. Relative expression level was computed using 2-ΔΔCt method.
For the data of each sample, the Ct value of the target gene was normalized to that of U6 (…).
The expression level of each gene was performed and the data were normalized to control unwanted sources of variation.
The relative expression level of indicated genes was compared with that of… and expression fold changes were calculated using 2-△△Ct methods.
第六句（可写可不写）
Each qRT-PCR reaction was performed in triplicate as follows: step 1: denaturation at 95°C for 10 min; step 2: 40 cycles of 95°C for 15 s and 60°C for 1 min.
PCRs were a relative estimation in triplicate as per the following temperature profile: denaturation 95 °C for 10 min followed by amplification by 40 cycles of 95 °C for 10 s and 60 °C for 1 min.
第七句（可写可不写）The cDNA was then diluted to 70 μL in water. qPCR was performed with 2×SensiFAST SYBR No-ROX kit (Bioline), gene-specific primers (250 nM final concentration of forward and reverse), and 2 μL cDNA. 举例
1. Total RNA was extracted using a Trizol reagent (Invitrogen). RNA (2 μg) was converted into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo). QRT-PCR was accomplished using the FastStart Universal SYBR Green Master (Rox) (Roche) in the ABI PRISM® 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA). GADPH and U6 were used as endogenous controls. We used melting curves to monitor non-specific amplifications. Relative expression level was computed using 2-ΔΔCt method. The primer sequences used are listed in Supplemental Experimental Procedures.
2. Total RNA was extracted by TRIzol reagent as described previously xx. The expression levels of 7 randomly selected differentially expressing circRNAs (Fold changes ≥ 2, p < 0.05) were measured by qRT-PCR; among them, 2 were upregulated and 5 were downregulated in the GC tissues: (upregulated: hsa_circ_0081146, hsa_circ_0084720), (downregulated: hsa_circ_0060108, hsa_circ_0057104, hsa_circ_0054971, hsa_circ_0063561, and hsa_circ_0058766). GAPDH expression was used as an internal reference. The primers used for these amplifications are listed in Table S1. PCRs were a relative estimation in triplicate as per the following temperature profile: denaturation 95 °C for 10 min followed by amplification by 40 cycles of 95 °C for 10 s and 60 °C for 1 min.
3. Total mRNA was isolated using Trizol (Invitrogen) and 2 μg RNA was used to synthesize cDNA using the PrimeScript RTreagent kit (Takara, DRR037A) according to the manufacturer’s protocol. Real-time PCR was performed using FastStart Universal Probe Master Mix (Roche) and analyzed with a Stratagene Mx 3000P thermal cycler. Real-Time PCR primer sequences are listed in Supplementary information, Table S1.
4. Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions, reverse transcribed and subjected to real-time PCR as previously described. [XX] Expression data were normalized to the geometric mean of the housekeeping gene GAPDH to control the variability in expression levels and calculated as 2- [(CT of indicated genes) - (CT of GAPDH)], where CT represents the threshold cycle for each transcript.
5. The total miRNA samples were extracted from ASPC-1 cells, PANC-1 cells and tumor tissues using SanPrep Column microRNA Mini-Preps Kit (Sangon Biological Engineering Technology & Services Co., Ltd., China) following the manufacturer’s protocol. The purity of the extracted miRNA was determined, and the RT-PCR was carried out using a microRNA First Strand cDNA Synthesis Kit following the manufacturer’s instructions with a TC-512 PCR system (TECHNE, UK). Quantitative real-time PCR assay was performed with a MicroRNAs Quantitation PCR Kit (Sangon Biological Engineering Tech- nology & Services Co., Ltd., China) and ABI 7500 Real Time PCR System (Applied Biosystems, USA). For the data of each sample, the Ct value of the target gene was normalized to that of U6 (Sangon Biological Engineering Technology & Services Co., Ltd., China). The unknown template was calculated using the standard curve for quantitative analysis. Eventually, the expression level of each gene was performed and the data were normalized to control unwanted sources of variation.
6. The total RNA of the tissue samples was extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was converted from total RNA by using a Reverse Transcription Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with SYBR Green (Takara, Dalian, China),0 and the data collection was performed on the Applied Biosystems® 7500 Real-Time PCR Systems (Thermo Fisher Scientific) according to the manufacturer’s instructions. The primers were synthesized by Biosune (Shanghai, China). The relative expression level of indicated genes was compared with that of β-actin and expression fold changes were calculated using 2-△△Ct methods. Each qRT-PCR reaction was performed in triplicate. Sequences of primers used for qRT-PCR in this study are shown in Additional file 1: Table S2.
7. Total RNA was extracted from cell lines, using Trizol total RNA isolation reagent (Invitrogen), according to the manufacturer’s specifications and treated with Turbo DNase (Ambion). cDNA was synthesized from total RNA (0.5 mg) using random hexamers with TaqMan cDNA Reverse Transcription Kit (Applied Biosystems). Gene primers (Gabra3 F-5’- GACCACGCCCAACAAGCT-3’; R-5’-AGCATGAATTGTTAACCTCATTGTATAGA-3’; GAPDH-F-5’-GAAGGTGAAGGTCGGAGTCAAC; R-5’-CAGAGT- TAAAAGCAGCCCTGGT-3’) were designed using Primer Express v3.0 Software and real-time PCR was performed using SYBR Select Master Mix (Applied Bio- systems). All reactions were carried out on the 7500 Fast Real-Time PCR System (Applied Biosystem). The average of three independent analyses for each gene and sample was calculated using the DD threshold cycle (Ct) method and was normalized to the endogenous reference control gene GAPDH.
8. TRIzol reagent (Thermo Fisher Scientific) was used to extract RNA from samples. Then a total of 250 ng of RNA was used to perform reverse transcription with QuantiMir RT kit (System Biosciences, Palo Alto, CA, USA). The primers used were as follows: CISD2, 5’-GCAAGGTAGCCAAGAAGTGC-3’ (forward) and 5’-CCCAGTCCCTGAAAGCATTA-3’ (reverse); GAPDH, 5’-GCGAGATCGCACTCATCATCT-3’ (forward) and 5’-TCAGTGGTGGACCTGACC-3’ (reverse). CISD2 was amplified as follows: step 1: denaturation at 95°C for 10 min; step 2: 40 cycles of 95°C for 15 s and 60°C for 1 min. Results were calculated with 2−DDCT and normalized to GAPDH.
9. Total RNA was extracted from cultured MCF7, A549 and UACC903 cells using Trizol reagent (Takara, Shiga, Japan) according to the manufacturer’s instruction. Reverse transcription was carried out with 0.5 μg total RNA using the PrimerScript™ RT reagent kit (Takara, Shiga, Japan). After incubation for 1 h at 42 °C, the reactions were terminated by heating at 70 °C for 15 min. To analyze alternative splicing of exon 2 in the Bcl-x gene, 5′ primer to Bcl-x (5′-GAGGCAGGCGACGAGTTTGAA-3′) and 3′ primer (5′-TGGGAGGGTAGAGTGGATGGT-3′) were used for PCR amplification (30 cycles, 94 °C, 30s; 56 °C, 30s; 72 °C, 1 min) with 2xEasy Taq superMix (Transgen Biotech, China). The length of splicing variants of Bcl-xL and Bcl-xS are 460 bp and 271 bp respectively. To analyze alternative splicing of exon 3, 4, 5, 6 in the Caspase 9 gene, 5′ primer to Caspase 9 (5′-GCTCTTCCTTTGTTCATCTCC-3′) and 3′ primer (5′-CATCTGGCTCGGGGTTACTGC-3′) were used for PCR amplification (30 cycles, 94 °C, 30s; 54 °C, 30s; 72 °C, 1 min). The length of splicing variants of Caspase 9a and Caspase 9b are 742 bp and 292 bp, respectively. PCR products were separated and analyzed on agarose gels, with the bands of the Bcl-x and Caspase 9 splicing variants being confirmed by DNA sequencing.
10. Brain tissues from the ipsilateral SN were dissected at the indicated time points after LPS or PBS injection with capsaicin or vehicle, and total RNA was extracted in a single step using RNAzol B (Tel-Test, Friendswood, TX) following the instructions of the manufacturer. Total RNA was reverse transcribed into cDNA using AMV reverse transcriptase (Promega, Madison, WI) and random primers (Promega). The primer sequences used in this study were as follows: 5′-TGATGTTCCCATTAGACAGC-3′ (forward) and 5′-GAGGTGCTGATGTACCAG TT-3′ (reverse) for IL-1β (378 bp); 5′-ACACTCTATCACTGGCATCC-3′ (forward) and 5′-AAGGGACACCCTTTCACAT-3′ (reverse) for COX-2; 5′-GCAGAA TGTGACCATCATGG-3′ (forward) and 5′-ACAACCTTGGTGTTGAAGGC-3′ (reverse) for iNOS (557 bp); and 5′-TCCCTCAAGATTGTCAGCAA-3′ (forward) and 5′-AGATCCACAACGGATACATT-3′ (reverse) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 308 bp). The PCR amplification consisted of 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s (for IL-1β and GAPDH or 54 °C for 30 s for iNOS) and extension at 72 °C for 90 s. PCR products were separated by electrophoresis on 1.5% agarose gels, after which the gels were stained with ethidium bromide and photographed. For semiquantitative analyses, the photographs were scanned using a Computer Imaging Device and the accompanying software (Fujifilm, Tokyo, Japan).
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