RNA extraction and purification were done using an RNeasy mini kit (Qiagen, Valencia, CA, USA) as described in the manufacturer's protocol. The concentration of RNA was measured using a spectrophotometer (DU530, Beckman, Fullerton, CA, USA). The total RNA sample (2 µg/sample) using the SuperScript III First-Strand Synthesis System for reverse transcriptase (RT) polymerase chain reaction (PCR) kit (Invitrogen, Milan, Italy) were used in a 20 µL scale to generate cDNA. RNA was reverse-transcribed at the following conditions: 25 mM MgCl2, 10 mM dNTP mix, 10 × RT buffer, 0.1 M DTT, 200 U of SuperScript III (Invitrogen), 40U of RNaseOut, 50 µM oligo d(T) primers in a final volume of 20 µL. The reaction was run at 65℃ for 5 minutes and 50℃ for 50 minutes, and then the enzyme was heat inactivated at 85℃ for 5 minutes. Four microliter of reaction products were used for real-time PCR reaction.
Polymerase chain reaction kit
Manufactured by Thermo Fisher Scientific
Sourced in Italy, United States
The polymerase chain reaction (PCR) kit is a laboratory tool used for the amplification of specific DNA sequences. It serves as a core function in various molecular biology applications, such as gene expression analysis, genetic profiling, and disease diagnosis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Du 530, by Beckman Coulter (1 mentions)
Superscript 3 first strand synthesis system for reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Sw480, by National Collection of Authenticated Cell Cultures (1 mentions)
2 protocols using polymerase chain reaction kit
1
RNA Extraction, Reverse Transcription, and Real-Time PCR
2
Culturing Human Cancer and Endothelial Cells
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Human colon cancer cell line SW480 was purchased from Cell Bank of the Chinese Academy of Science, Shanghai, China. SW480 and human umbilical vein endothelial cells (HUVEC) were cultured in RPMI 1640 medium containing penicillin, streptomycin, and 10% fetal bovine serum (FBS) at 37°C in an incubator with 5% CO 2 . RPMI 1640 medium, FBS, and trypsin were purchased from Gibco (Carlsbad, CA, USA). Reverse transcription kit was purchased from TaKaRa (Dalian, China). Polymerase chain reaction (PCR) kit was purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against FLT-1, VE-cadherin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Abcam (Cambridge, MA, USA), and pBLAST49-hsFLT-1 recombinant plasmid and pBLAST49 were purchased from Invitrogen (Carlsbad, CA, USA). Matrigel gum was purchased from BD Biosciences (San Diego, CA, USA).
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