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Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

BS Bin Shen
LW Lan Wang
YX Yuejun Xu
HW Hongwei Wang
SH Shiyi He
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Reverse transcription was carried out with miRcute miRNA First-Strand complementary DNA (cDNA) Synthesis Kit (for miR-34b-3p; TIANGEN, Beijing, China) and TaqMan reverse transcription kit (for GAS5; Applied Biosystems, Rotkreuz, Switzerland). cDNA was amplified with TransStarts Green qPCR SuperMix (Transgen, Beijing, China). U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the internal references for miR-34b-3p and GAS5, respectively. The primers for GAS5, miR-34b-3p and internal controls were listed as follows. Mouse GAS5, Forward: 5ʹ-GGATAACAGAGCGAGCGCAAT-3ʹ, Reverse: 5ʹ-CCAGCCAAATGAACAAGCATG-3ʹ. Mouse miR-34b-3p, Forward: 5ʹ-AATCACTAACTCCACTGCCATC-3ʹ, Reverse: 5ʹ-GATGGCAGTGGAGTTAGTGATT-3ʹ. Mouse U6, Forward: 5ʹ-CGCTTCGGCAGCACATATACTA-3ʹ, Reverse: 5ʹ-CGCTTCACGAATTTGCGTGTCA-3ʹ. Mouse GAPDH, Forward: 5ʹ-GGAGCGAGACCCCACTAACAT-3ʹ, Reverse: 5ʹ-GTGAGTTGTCATATTTCTCGTGG-3ʹ.

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