Ncode mirna universal qpcr primer

Total RNA was isolated from cells with the TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using an M-MLV Reverse Transcriptase Kit (Invitrogen). Quantitative real-time PCR was carried out using SYBR Premix Ex Taq II (Takara, Shiga, Japan) in an ABI-7300 Real-Time PCR system (Applied Biosystems, Foster City, CA). The primers were as follows: human GLUT1, sense: 5′-GGCCAAGAGTGTGCTAAAGAA-3′, anti-sense: 5′-ACAGCGTTGATGCCAGACAG-3′; human MMP-1, sense: 5′-AAAATTACACGCCAGATTTGCC-3′, anti-sense: 5′-GGTGTGACATTACTCCAGAGTTG-3′; human MMP-14, sense: 5′-GGCTACAGCAATATGGCTACC-3′, anti-sense: 5′-GATGGCCGCTGAGAGTGAC-3′; human Cyclin D1, sense: 5′-GCTGCGAAGTGGAAACCATC-3′, anti-sense: 5′-CCTCCTTCTGCACACATTTGAA-3′; human TMPO-AS1, sense: 5′-GTGCTGCAGGACCGAGG-3′, anti-sense: 5′-GCTTTGTGTCCGCGAGTTTT-3′, and human β-actin, sense: 5′-CATGTACGTTGCTATCCAGGC-3′, anti-sense: 5′-CTCCTTAATGTCACGCACGAT-3′. Gene expression was normalized to beta-actin mRNA. The expression of miR-140 and miR-143 was measured using the NCode SYBR GreenER miRNA qRT-PCR analysis kit (Invitrogen). The forward primers for miRNA analysis had the same sequences as the mature miRNAs. The forward primer for U6 was 5′-CGCAAGGATGACACGCAAATTCG-3′. The reverse primer was the NCode miRNA universal qPCR primer (Invitrogen). The expression of miRNAs was calculated relative to U6.

An NCode SYBR green miRNA qRT-PCR Kit (Invitrogen) was used to validate miR-374a expression. Briefly, 200 ng of small RNA was converted to cDNA. The reverse primer was the NCode miRNA universal qPCR primer (Invitrogen). The forward primer for miR-374a was 5′-TAATACTGCCGGGTAATGATGG-3′; the cycle threshold (Ct) was recorded. The expression of miR-374a in tissues was calculated relative to U6B, a ubiquitously expressed small nuclear RNA that has been widely used as an internal control (32 (link)). Data are presented as miR-374a expression = 2Δ^Ct, with ΔCt = (U6B Ct − miR-374a Ct).