Quantitative reverse transcription PCR (qRT-PCR) Total RNA was extracted by TRIzol (Invitrogen) and 5 μg RNA was used to generate complementary DNA. Transcriptional levels of genes were determined by Premix
2×Taq plus Master Mix专为常规PCR扩增反应优化,使用时只需再加入模板和引物并用水补足体系至反应浓度为1×,即可进行PCR反应,大大地简化了操作过程,减少了PCR操作过程中的污染。经测试,染料的加入不影响PCR反应,在PCR反应完成后可直接电泳,节省时间。使用本试剂扩增得到的PCR产物3´端有一突出“A”碱基,可直接...
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, complementary DNA (cDNA) was synthesized using a PrimeScriptTMRT Master Mix Kit (TaKaRa, Dalian, China). Real-time PCR analysis was performed using ...
Freezing and thawing were repeated two more times to lyse the cells, and 2 units of recombinant DNase I (Takara Bio) was added to the solution. The solution was incubated at room temperature until the solution became fluid due to DNA degradation. The solution was then centrifuged at 12 000...
RT–PCR was performed to detect sgRNA3 and GAPDH mRNA by using Quick Taq HS Dye Mix (Toyobo). Quantitative RT–PCR was performed by using Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and a StepOne Plus Real-Time PCR System (Applied BioSystems). The expression level ...
cDNA制备:根据PrimeScriptTMRTMasterMix说明书操作,反应体系为 5×PrimeScriptRTMasterMix4µL,16μL浓度为50ng/μL的RNA,充分混匀、瞬 PCR37℃15min85℃5s4℃-20℃ 离后放于仪;反应程序为,,,降温至,可 保存。 PCRTBGreen®PremixExTaq™IITakaracDNA 荧光定量:使用()对制备的 进行RealTime-PCR实验。R...
aqRT-PCR analysis ofHDAC2-AS2expression relative to the spike-in control λ polyA+RNA in the TCS of MHCC97L and MHCC97H cells treated with 2 μg/mL RNase A alone or in combination with 0.1% Triton X-100 for 20 min.bRepresentative electron microscopy images of EVs. Scale bar: 100...
RNA preparation and quantitative real-time PCR Total RNA was extracted according to the manufacturer’s instructions. RT-qPCR was performed using a SYBR Premix Ex Taq (TaKaRa) on a Thermal Cycler Dice Real Time System (TaKaRa) with the following protocol: 30 s at 95°C followed by two-step...
MA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Complementary DNA samples were diluted 1.5-fold, and qRT-PCT was performed using an ABI-7500 Real-Time PCR System (Applied Biosystems, Waltham, MA) with SYBR Premix Ex-Taq II (Takara Bio Inc., Shiga, Ja...
Total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara Bio, Kusatsu, Japan). This was followed by real-time quantification using GoTaq qPCR Master Mix (Promega, Madison, WI, USA). The PCR control we used was the housekeeping gene glyceraldehyde-3-phosphate ...