RNA was extracted from HAECs using TRIzol. The concentration of RNA was assessed using a NanoDrop 2000c spectrophotometer. The reverse transcription system included 5 × RT master Mix (2 µl, TOYOBO, Japan), RNA template (1 µg), and RNase-free water in a final volume of 10 µl. The reaction conditions were as follows: 37 °C for 15 min, 50 °C for 5 min, and 98 °C for 5 min. Real-time PCR was performed using a SYBR probe (Applied Biosystems, USA). An ABI 7900HT (Applied Biosystems, USA) thermocycler was used for qPCR amplifications. Relative expression (fold change vs. control) was calculated with the 2-ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as the reference. The primer sequences used are shown in Supplemental Table 2 .
Sybr probe
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYBR probe is a fluorescent dye used for detecting and quantifying DNA in real-time PCR experiments. It binds to double-stranded DNA, emitting a fluorescent signal that can be measured to determine the amount of DNA present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rt master mix, by Toyobo (1 mentions)
Abi 7900ht, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by CWBIO (1 mentions)
Quantstudio 6flex qrt pcr, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hiscript q rt supermix kit, by Vazyme (1 mentions)
2 protocols using sybr probe
1
RNA Extraction and Quantitative RT-PCR
2
Quantitative Analysis of AML Samples
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Bone marrow samples were obtained from 19 AML patients and 19 healthy donors from the First Hospital of Wenzhou Medical University. Our study was approved by the ethics committee of the First Hospital of Wenzhou Medical University1.
Quantitative real-time PCR (qRT-PCR) total RNA was extracted using TRIZOL reagent (Life Technologies). reverse transcription was then performed using the HiScript Q RT SuperMix kit (Vazyme, Nanjing, China). qRT-PCR was performed on an Applied Biosystems Quantstudio 6Flex qRT-PCR using a SYBR probe (Applied Biosystems, Foster City, CA, USA). then, qRT-PCR was performed to assess mRNA expression using SYBR Green Master Mix (CWBIO, Jiangsu, China) in an Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) as previously described.
Quantitative real-time PCR (qRT-PCR) total RNA was extracted using TRIZOL reagent (Life Technologies). reverse transcription was then performed using the HiScript Q RT SuperMix kit (Vazyme, Nanjing, China). qRT-PCR was performed on an Applied Biosystems Quantstudio 6Flex qRT-PCR using a SYBR probe (Applied Biosystems, Foster City, CA, USA). then, qRT-PCR was performed to assess mRNA expression using SYBR Green Master Mix (CWBIO, Jiangsu, China) in an Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) as previously described.
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