The qRT-PCR experiment is described in previous reports.25 (link) In brief, the total RNA of HPMECs/HBECs was isolated by TRIzol reagent (Invitrogen). The PrimeScipt RT Master Mix kit (Takara, Tokyo, Japan) was used to prepare cDNA. The SYBR® Premix Ex TaqTM reagent (TaKaRa) was used to quantitatively analyze gene expression. The 2−∆∆Ct method was used to calculate the relative expression of MEG3/miR-421/DFFB. GAPDH and U6 snRNA served as the normalized controls. The primers for MEG3, miR-421, and DFFB were purchased from GenePharma, and the specific sequences were as followed: U6: F: 5’- CTCGCTTCGGCAGCACA-3’, R: 5’- AACGCTTCACGAATTTGCGT-3’. MEG3: F: 5’-CAGGATGGCAAAGGATGAAG-3’, R: 5’-GCAGGTGAACACAAGCAAAGA-3’. miR-421: F: 5’- GTCGCGCGGGUUAAUGCCTC-3’, R: 5’- GGACATUAGUUGUCUGUAAATAG-3’. DFFB: F: 5’- CACAACGTCAGCCAGAACAT-3’, R: 5’- CCCAGTCCACTTCTCTTCCA-3’. GAPDH: F: 5’-TGTTCGTCATGGGTGTGAAC-3’, R: 5’- ATGGCATGGACTGTGGTCAT-3’.
Primescipt rt master mix kit
Manufactured by Takara Bio
Sourced in Japan, China
The PrimeScipt RT Master Mix kit is a versatile reagent used for reverse transcription (RT) of RNA to complementary DNA (cDNA). It contains all the necessary components, including reverse transcriptase, RNase inhibitor, and buffer, to efficiently convert RNA into cDNA in a single-tube reaction.
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Lab products found in correlation
2 protocols using primescipt rt master mix kit
1
Gene Expression Analysis of MEG3, miR-421, and DFFB
2
Cartilage RNA Extraction and Quantification
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Total RNA from cartilage tissues and primary chondrocytes was extracted by using TRIzol reagent (Invitrogen: Carlsbad, CA, USA). Total RNA was reverse‐transcribed into cDNA with the PrimeScipt RT Master Mix Kit (Takara: Dalian, China) and SYBR PrimeScript miRNA RT‐PCR Kit (Takara: Dalian, China). cDNA levels were monitored by qRT‐PCR analysis on a 7500 Sequence Detection System (Applied Biosystems: Foster City, CA, USA) using gene‐specific primers (available when requested) and SYBR Premix Ex Taq (Takara: Dalian, China). Fold expression change was calculated by the comparative threshold cycle (Ct) with the formula 2−ΔΔCt method. RNU6B and GAPDH were measured as endogenous controls for miRNA and mRNA, respectively.
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