LncRNA expression from the RNA sequencing data analysis was validated by qRT-PCR. A fixed volume of total RNA was subjected to reverse transcription using a PrimeScript RT Reagent Kit (Takara-Bio, Dalian, China) with a gDNA Eraser in accordance with the manufacturer’s protocol. The Premix Ex Taq II Reagent Kit (Takara-Bio, Dalian, China) and ABI 7500 Real-Time PCR thermocycler (Applied Biosystems, Carlsbad, CA, USA) were used for the qRT-PCR reactions. The primer sequences were listed in Table S1 . Each reaction was conducted in triplicate. LncRNA expression was calculated using cycle threshold (Ct) values, and was internally normalized to glyceraldehyde 3-phosphate dehydrogenase using the 2−ΔΔCT equation method. All primer pairs are available upon request.
Premix ex taq 2 reagent kit
Manufactured by Takara Bio
Sourced in China, Japan
Premix Ex Taq II Reagent Kit is a ready-to-use PCR reagent solution that contains all the necessary components for efficient DNA amplification. It is suitable for a wide range of PCR applications.
Automatically generated - may contain errors
2 protocols using premix ex taq 2 reagent kit
1
Validation of LncRNA Expression by qRT-PCR
2
Quantitative Real-Time PCR Analysis
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Total RNA from cells was extracted using a Tiangen Total RNA Extraction Kit (Tiangen, China, DP424). The concentration and purity of total RNA were detected after extraction. Reverse transcription of 500 ng RNA into cDNA was performed according to the instructions of the TAKARA Reverse Transcription Kit (TaKaRa, Japan, RR047A). SYBR was detected according to the instructions of the ® Premix Ex Taq™ II reagent kit (TaKaRa, Japan, RR820A) in a CFX96 Real Time PCR Detection System (Bio Rad) instrument. The following reaction conditions were used: 95℃ for 30 s; 40 cycles at 95℃ for 10 s, 56℃ for 30 s and 72℃ for 30 s; 72℃ for 5 min. Each sample was inserted into 3 holes, and the experiment was repeated 3 times. The 2-ΔΔCt method was used to analyze and process the data (See Table 2 of Supplementary Materials for specific primer sequences).
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