RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR)

YL Yibing Li
JH Jianing Huo
JH Junjian He
XM Xiaoxin Ma
request Request a Protocol
ask Ask a question
Favorite

Total RNA was extracted from tissues and cells using TRIzol reagent (Takara, Beijing, China). The complementary DNAs (cDNAs) for the LncRNAs and mRNAs of interest were reverse-transcribed from 2 μg total RNA using PrimeScript RT-polymerase (Takara). The cDNAs for the miRNAs of interest were synthesized from 1 μg total RNA using miRNA 1st Strand cDNA Synthesis SuperMix (Vazyme, Nanjing, China). We performed qRT-PCR using SYBR-Green Premix (Takara), miRNA Universal SYBR® qPCR Master Mix (Vazyme), and specific PCR primers (Sangon Biotech, Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were used as internal controls. We determined the expression of LncRNA, mRNA, and miRNA using the calculating 2−ΔΔCT method. Primer sequences are summarized in Table Table11.

Primer Sequences

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A