Q6 real time pcr system

Total RNA was isolated from tissue samples or cultured cells using Qiazol reagent (Qiagen, Gaithersburg, MD, USA) or TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse‐transcribed into miRNA cDNA or total cDNA using the Prime Script miRNA cDNA synthesis kit (Qiagen) or PrimeScript RT kit (Qiagen), and then subjected to quantitative RT‐PCR (qRT‐PCR). mRNA or miRNA expression was determined by qPCR on a Q6 real‐time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR Green Master Mix (4309155, Applied Biosystems) or the miScript PCR Kit (Qiagen), using GAPDH or U6, respectively, as the internal loading control. Reverse transcription and PCR were performed as described in our previous report.⁵⁵ Specific primers for human MMP9 (Forward: 5′‐TTC CAA ACC TTT GAG GGC GA‐3′ and Reverse: 5′‐CTG TAC ACG CGA GTG AAG GT‐3′), human SMURF1 (Forward: 5′‐AAC TGA AAC CCA ATG GCA GAA ATGT‐3′ and Reverse: 5′‐TTG CCA GAA CCA CCG CAC GAT G‐3′), and human GAPDH (Forward: 5′‐GAC TCA TGA CCA CAG TCC ATG C‐3′, Reverse: 5′‐CAG GTC AGG TCC ACC ACC ACT GA‐3′) were used for PCR amplification. The primer for miR‐516a (5′‐TGC TTC CTT TCA GAG GGT‐3′) was synthesized by Sunny Biotechnology (Shanghai, China), and U6 was used as the internal loading control through the primer provided in the miScript PCR Kit. The data were analyzed as previously described.⁵⁵

Total RNA from the cells was isolated using the trizol reagent. Total RNA (5 µg) was then used for reverse transcription with oligo dT primer through the SuperScript™ First-Strand Synthesis system IV (Invitrogen, Grand Island, NY). Specific primer pairs were designed to amplify human CDKN1B (forward: 5′-CAA GTA CGA GTG GCA AGA G-3′, reverse: 5′-ATG CGT GTC CTC AGA GTT AG -3′) and GAPDH (forward: 5′-AGA AGG CTG GGG CTC ATT TG-3′, reverse: 5′-AGG GGC CAT CCA CAG TCT TC-3′). Total miRNAs were extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA (1.0 µg) was used for reverse transcription following the manufacturer’s instructions, and miRNA expression was determined by the Q6 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using the miScript PCR Starter Kit and miScript PCR kit II RT Kit (Qiagen, Valencia, CA, USA). U6 was used as the endogenous normalizer. The primer for miR-190 (5′- TGA TAT GTT TGA TAT ATT AGG T-3′) was synthesized by Genewiz Biotechnology (South Plainfield, USA). The cycle threshold (CT) value was measured, and the relative expression of mRNA was calculated based on the value of 2^−ΔΔCT as described in our published studies [21 (link)].

Total RNA was isolated from cultured cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse-transcribed into total cDNA using the PrimeScript^TM RT kit (#RR036A; TaKaRa, Kyoto, Japan) and then subjected to RT-PCR. mRNA expression levels were determined by qPCR on a Q6 real-time PCR System (Applied Biosystems, Carlsbad, CA, USA) with SYBR qPCR Master Mix (4309155, Applied Biosystems) using GAPDH as an internal loading control. The primers used in this study were as follows: human Akt1 (forward: 5′-CTTGCTTTCAGGGCTGCTCA-3′ and reverse: 5′-TACACGTGCTGCCACAGGA TAC-3′); human STUB1 (forward: 5′-CTCTCACGCTCCGCGGCAAT-3′ and reverse: 5′-GCCAAGGAGCAGCGGCTGAA-3′); and human GAPDH (forward: 5′-GACTC ATGACCACAGTCCATGC-3′ and reverse: 5′-CAGGTCAGGTCCACCACTGA-3′).

Total RNA was isolated from samples using a plant RNA purification kit (Tiangen). The first-strand cDNA was synthesized using the PrimeScript™ 1st Strand cDNA Synthesis Kit for RT-PCR (TaKaRa). Transcript levels were determined by quantitative real-time PCR (qRT-PCR) analysis using the Q6 Real-Time PCR System (Applied Biosystems) and SYBR Premix Ex Taq (2×) (TaKaRa). To normalize these samples, GhACTIN was as an endogenous control. Determination of reaction specificities and data processing were performed as described in previous study (Schmittgen and Livak, 2008 (link)). Gene-specific primers used for the PCR are listed in Supplementary Table 1. Three biological replicates were analyzed. The significance of differences between means was determined using analysis of variance implemented in SAS software (^∗P < 0.05, ^∗∗P < 0.01). The data were graphed using GraphPad Prism 5.

First-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) with total RNA as template. Quantitative RT-PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme) in triplicate on the Q6 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative quantification was performed using the 2^−ΔCt method, and the ACTIN gene (Zm00001d010159) was used as control. The primers used in qRT-PCR analysis are listed in Table S6.