RNA was isolated from HMEC-1 treated with PBS or E. coli OMVs (0.5 ng/mL) for 12 h, using the RNeasy Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s protocol. Total RNA (100 ng in total amounts) was subject to amplification with the One Step SYBR RT-PCR Kit (Takara Bio Inc., Kusatsu, Japan) using the LightCycler 2.0 PCR System (Roche Diagnostics, Basel, Switzerland). Primers were designed using the Primer3 program (Untergasser et al., 2012 (link)), and the primers used for real-time RT-PCR are shown in Supplementary Table 1 . Amplification was performed by heating the samples at 50°C for 2 min, then at 95°C for 10 min, followed by repeating cycles of 95°C for 15 s, 55°C for 10 s, and 72°C for 10 s, for a total of 45 cycles. The comparative Ct method was employed to relatively quantify the expression of target genes against that of a house-keeping gene GAPDH (Livak and Schmittgen, 2001 (link)).
Lightcycler 2.0 pcr system
Manufactured by Roche
Sourced in Japan
The LightCycler 2.0 PCR system is a real-time PCR instrument designed for nucleic acid amplification and detection. It is capable of performing quantitative, qualitative, and melting curve analyses. The system utilizes fluorescence-based detection methods to monitor the progress of PCR reactions in real-time.
Automatically generated - may contain errors
Lab products found in correlation
One step sybr rt pcr kit, by Takara Bio (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Superscript 2 kit, by Thermo Fisher Scientific (2 mentions)
Sybr green mix, by Thermo Fisher Scientific (2 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Promega (1 mentions)
Rnase easy columns, by Qiagen (1 mentions)
6 protocols using lightcycler 2.0 pcr system
1
RNA Isolation and Quantification of Gene Expression
2
Strand-Specific RT-PCR Protocol
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RNA was purified as described for microarray analysis and subjected to RT–PCR using a One Step RT‐PCR kit (Qiagen). For strand‐specific RT–PCR, one primer complementary to the sense or antisense transcript was added during first‐strand cDNA synthesis, while the second primer was added prior to the PCR amplification steps. cDNA for quantitative (real‐time) RT–PCR was made using a Superscript II kit (Invitrogen). Real‐time PCRs were performed using a LightCycler 2.0 PCR system (Roche) and SYBR Green mix (Molecular Probes) using the appropriate primers. Reactions were normalised using primers specific to act1+. Northern analysis was performed as previously described 65 .
3
Quantitative Analysis of miRNA and mRNA
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Total RNA was isolated from cells and tissues using TRIzol reagent. Nuclear and cytoplasmic components were separated using the PARIS Kit according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification was carried out by a two-step method, the cycle parameters were: pre-denaturation at 95 °C for 30 s, then 40 cycles of: 95 °C for 5 s, 56 °C for 20 s, and finally melting curve analysis. The entire PCR reaction and analysis were performed in the LightCycler 2.0 PCR system (Roche, Basel, Switzerland). We analyzed the fluorescence signal in the amplification and melting curves, and the average cycle threshold (Ct) values were calculated, followed by calculation of the relative expressions of the target genes by the 2−ΔΔCt method. The relative gene expressions of miR-200c-3p and miR-141-3p were calculated with U6. Bulge-loopTM miRNA qRT-PCR Primer Sets specific for miR-200c-3p, miR-141-3p, and U6 were purchased from Ribobio (Guangzhou, China). Relative gene expressions of MIR200CHG and YB-1 mRNA were calculated with GAPDH. The primer sequences of MIR200CHG, YB-1, and GAPDH are listed in Supplementary Note 2 .
4
Real-time RT-PCR for Gene Expression
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PCR primers used in this study were designed using the Primer3 program (Whitehead Institute, http://biotools.umassmed.edu/bioapps/primer3_www.cgi ). The primers used for gene amplification were as follows: GAPDH forward, 5′-CGAGATCCCTCCAAAATCAA-3′ ; GAPDH reverse, 5′-TTCACACCCATGACGAACAT-3′ ; EGR1 forward, 5′-CCGCAGAGTCTTTTCCTGAC-3′ ; EGR1 reverse, 5′-AGCGGCCAGTATAGGTGATG-3′ . HMEC-1s (2×105 cells) plated in 6-well cell-culture plate were treated with EVs (1 µg/mL, 2.0 mL) for 0.5, 1, 1.5, 2 and 4 h. RNA was extracted from cultured cells using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). For real time RT-PCR, total RNA (100 ng) was amplified with a One Step SYBR RT-PCR Kit (TaKaRa Bio, Tokyo, Japan) using a LightCycler 2.0 PCR system (Roche Diagnostics, Mannheim, Germany). Amplification was carried out by heating the samples to 50°C for 2 min, then at 95°C for 10 min, followed by repeating cycles at 95°C for 15 sec, 55°C for 10 sec, and 72°C for 10 sec, for a total 45 cycles. The comparative Ct method was used for relative quantification of target gene expression against that of a housekeeping gene, GAPDH [40] (link).
5
Gene Expression Analysis of hAMSCs Differentiation
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Total RNA was extracted from hAMSCs using TRIzol reagent (Invitrogen, USA). cDNA was reverse-transcribed from 1.5 μg total cellular RNA using oligo(dT) primers and Moloney Murine Leukemia Virus reverse transcriptase (Promega Corp., USA). cDNA was amplified using the following primers: peroxisome proliferator-activated receptor gamma (PPAR-γ); CCAAT-enhancer-binding proteins (C/EBP)-α 5′-AACCTTGTGCCTTGGAAATG-3′ (sense), 5′-CCTGCTCCCCTCCTTCTCT-3′ (antisense); adiponectin 5′-ACCACTATGATGGCTCCACT-3′ (sense), 5′-GGTGAAGAGCATAGCCTTGT-3′ (antisense); fatty acid binding protein (aP2) 5′-AACCTTAGATGGGGGTGTCCTG-3′ (sense), 5′-TCGTGGAAGTGACGCCTTTC-3′ (antisense); lipoprotein lipase (LPL) 5′-CTGGACGGTAACAGGAATGTATGAG-3′ (sense), 5′-CATCAGGAGAAAGACGACTCGG-3′ (antisense), and the reference gene, ribosomal protein large P0 (RPLP0) 5′-GGAATGTGGGCTTTGTGTTC-3′ (sense), 5′-TGCCCCTGGAGATTTTAGTG-3′ (antisense). For real-time PCR, total RNA (100 ng) was amplified with One Step SYBR RT-PCR kit using a Light Cycler 2.0 PCR system (Roche Diagnostics, Manheim, Germany). PCR conditions consisted of a 10-min hot start at 95°C, followed by 45 cycles of 15 s at 95°C, 10 s at 60°C, and 30 s at 72°C. mRNA expression levels were normalized against RPLP0 expression. The experiment was repeated twice.
6
RNA Extraction and Strand-Specific RT-qPCR
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RNA was extracted using hot phenol and purified over RNase easy columns (QIAGEN, Valencia, CA) as previously described (Lyne et al. 2003 (link)). For strand-specific RT-PCR, one primer complementary to the sense or antisense transcript was added during first strand cDNA synthesis, while the second primer was added prior to the PCR amplification steps. cDNA for quantitative PCR (qPCR) was made using a Superscript II kit (Invitrogen, Carlsbad, CA). qPCR reactions were performed using a LightCycler 2.0 PCR system (Roche Diagnostics, Indianapolis, IN) and SYBR Green mix (Molecular Probes, Eugene, OR) using the appropriate primers.
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