RNA was isolated from cultured cells or MV samples using an RNeasy Mini Kit (Qiagen). Total RNA (1 μg) was reverse transcribed using an RT-PCR Kit (TOYOBO). Complementary DNA (cDNA) was analyzed using a GeneAmp 7500 Fast Real-Time PCR System (Life Technologies) using the SYBR Green reagent (TOYOBO). The expression levels of the target genes were analyzed with the ΔΔCt method. The primers used for the polymerase chain reaction (PCR) were as follows: VEGF (5′-AGATGAGCTTCCTACAGCACAAC; 3′-AGGACTTATACCGGGATTTCTTG), CXCR4 (5′-CTGTGACCGCTTCTACCCCAATGACTT; 3′-CCAAGGAAAGCATAGAGGATGGGGTTC), KDR (5′-AGTGTGGAGGACTTCCAGGGAGGAAAT; 3′-GGCCAAGCTTGTACCATGTGAGGTTCT), SDF-1 (5′-TGAGAGCTCGCTTTGAGTGA; 3′-CACCAGGACCTTCTGTGGAT), VCAM-1 (5′-GTAAGCTGCAAGGTTCCTAGCGTGT; 3′-GCTGACCAAGACGGTTGTATCTCTG), Glut-1 (5′-ACTGCTCAAGAAGACATGGAGAC; 3′-ATTTACAAGTTGGCTTGTCCAGA), TGF-β (5′-AGAGCTCCGAGAAGCGGTACCTGAACCC; 3′-GTTGATGTCCACTTGCAGTGTGTTATCC), HIF-1α (5′-TTACCGAATTGATGGGATATGAG; 3′-TCATGATGAGTTTTGGTCAGATG), Col IV (5′-AGGGCCAGCCTGGCCTGCCAGGACTTCC; 3′-TCACCCTTAGAGCCTGTGATTCCTGGAG), and β-actin (5′-GTGCGTGACATTAAGGAGAAGCTGTGC; 3′-GTACTTGCGCTCAGGAGGAGCAATGAT).
Geneamp 7500 fast real time pcr system
Manufactured by Thermo Fisher Scientific
The GeneAmp 7500 Fast Real-Time PCR System is a high-performance thermal cycler designed for conducting real-time PCR experiments. It provides accurate temperature control and rapid thermal cycling capabilities to support a wide range of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Rt pcr kit, by Toyobo (3 mentions)
Sybr green reagent, by Toyobo (2 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
Rnu48, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Amperase ung, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix 2, by Thermo Fisher Scientific (1 mentions)
Isogen ls, by Nippon Gene (1 mentions)
4 protocols using geneamp 7500 fast real time pcr system
1
Quantitative gene expression analysis
2
Quantitative PCR Analysis of AT-MSCs
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Total RNA from AT-MSCs was isolated using Sepasol-RNA I Super G (Nacalai Testque, Kyoto, Japan) according to the previous described method44 (link). In order to synthesize cDNA, a RT-PCR kit (Toyobo Co., Ltd, Osaka, Japan) was used with 1 µg of total RNA. Next, the expression of the target genes was examined by quantitative PCR of cDNA. The reaction mixtures for qPCR were prepared using SYBRGreen Realtime PCR mastermix (Toyobo) and analyzed using a GeneAmp 7500Fast Realtime PCR System (Life Technologies). The sequences of primers used for the PCR reactions are listed in Table 1 . The experiments were performed in triplicate, and data were calculated by the double-delta cycle number of thresholds (ΔΔCt) method.
Primer sets used for quantitative PCR.
| Gene | Forward primer | Reverse primer |
|---|---|---|
| il6 | ACAAGAGTAACATGTGTGAAAGCAG | TATACCTCAAACTCCAAAAGACCAG |
| il8 | TGCTTCCCCTTAGCATTTTGT | TGTCCAGCTATGCTAAAGTGC |
| ccl5 | GAGGATTCCTGCAGAGGATCAAGACAG | TCCAAAGAGTTGATGTACTCCCGAACC |
| ccl3 | CAGCCTGTGTAGGCAGTCAT | CTCCCCATCTCTCCCAATTTCC |
| vegf | AGATGAGCTTCCTACAGCACAAC | AGGACTTATACCGGGATTTCTTG |
| ang1 | GCCTGATCTTACACGGTGCT | GGCCACAAGCATCAAACCAC |
| sdf1 | AGAGCCAACGTCAAGCATCT | CTTTAGCTTCGGGTCAATGC |
| bfgf | AGAGCGACCCTCACATCAAGCTACAAC | ATAGCTTTCTGCCCAGGTCCTGTTTTG |
| flk1 | AGTGTGGAGGACTTCCAGGGAGGAAAT | GGCCAAGCTTGTACCATGTGAGGTTCT |
| β-actin | GTGCGTGACATTAAGGAGAAGCTGTGC | GTACTTGCGCTCCAGGAGGAGCAATGAT |
3
Quantifying Gene Expression in Calu-3 Cells
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To examine the gene expression, total RNA was collected from Calu-3 cells and isolated by Sepasol-RNA I Super G (Nacalai Tesque) in accordance with the manufacturer's protocol. Total RNA (1 μg) was reverse transcribed using an RT-PCR kit (Toyobo, Osaka, Japan). cDNA was analyzed using a GeneAmp 7500Fast Real-Time PCR System (Applied Biosystems) using SYBR green reagent (Toyobo). The expression levels of the target genes were analyzed using the ΔΔCt method. The sequences of the primer sets used for the PCR are shown in Table 1 .
4
Quantitative RT-PCR Analysis of RNA
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Total RNA of cell samples were isolated by Sepasol-RNA I Super G (Nacalai Tesque) in accordance with the manufacturer’s protocol and the total RNA of EV samples were isolated using ISOGEN-LS (Nippon gene, Tokyo, Japan) in accordance with the manufacturer’s protocol. Total RNA (1 µg) was reverse transcribed using a TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). cDNA (500 ng) was analyzed using a GeneAmp 7500Fast Real-Time PCR System (Applied Biosystems) using TaqMan 2 × Universal PCR Master Mix, with AmpErase UNG (Applied Biosystems). The RNU48 was used as internal control (ThermoFisher). The expression levels of the target genes were analyzed using the ΔΔCt method. The sequences of the primer sets used for PCR are shown in Table 1 .
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