Ssrt 4 system

Trizol reagent (Invitrogen, USA) was used to extract total RNAs. RNA precipitation and washing were performed using 85% ethanol to retain miRNAs. Genomic DNA in RNA samples was removed using LookOut^® DNA Erase (Sigma-Aldrich, USA). The SSRT IV system (Invitrogen, USA) was used to perform reverse transcriptions (RTs) with poly (T) as primer. The qPCR mixtures were prepared using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent, USA). Expression levels of GAS5 were normalized to GAPDH endogenous control. For the quantification of miR-21, All-in-One^TM miRNA qRT-PCR Detection Kit (GeneCopoeia, Shanghai, China) was used to perform polyadenylation, RTs and qPCR assays. Expression levels of miR-21 were normalized to U6 endogenous control. Primer sequences were: 5ʹ-ACACTCCAGCTGGGTAGCTTATCAG-3ʹ (forward) and 5ʹ-TGGTGTCGTGGAGTCG-3ʹ (reverse) for miR-21; 5ʹ-CTTCTGGGCTCAAGTGATCCT-3ʹ (forward) and 5ʹ-TTGTGCCATGAGACTCCATCAG-3ʹ (reverse) for miR-21; 5ʹ-AAGGTGAAGGTCGGAGTCAA-3ʹ (forward) and 5ʹ-ATGAAGGGGTCATTGATGG-3ʹ (reverse) for GAPDH; 5ʹ-GCTTCGGCAGCACATATACTAAAATTGGA-3ʹ (forward) and 5ʹ-CTTCACGAATTTGCGTGTCATCCTTG-3ʹ (reverse) for U6. Three replications were set for each experiment. PCR reaction conditions were: 95°C for 1 min, followed by 40 cycles of 9 5°C for 10 s and 55°C for 45 s. 2^−ΔΔCt method was used to calculate the fold changes of gene expression across samples.

All RNA extractions in this study were performed using Trizol (Invitrogen, USA). DNA eraser (Sigma-Aldrich) was used to remove genomic DNAs from all RNA samples. After that, 20 μL RT reaction systems were prepared using SSRT IV system (Invitrogen, USA) to transcribe total RNA into cDNA. Reaction thermal conditions were: 30 min at 45°C and 10 at 55°C. To measure the levels of PTCSC3 and HOXA11-AS expression, 2 × SYBR^® Premix Ex Taq™ (Takara) was used to prepare qPCR reaction mixtures with cDNA as template and gene-specific primers. Eppendorf Mastercycler (Eppendorf, Germany) was used to carry out all qPCR reactions through following thermal conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 55°C for 40 s. Primers for PTCSC3, HOXA11-AS and endogenous control GAPDH were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The following primer sequences were used: PTCSC3 forward 5′-TCAAACTCCAGGGCTTGAAC-3′, and reverse 5′-ATT ACGGCTGGGTCTACCT-3′; HOXA11-AS forward, 5ʹ-AGCAACAGATCGT -CACTCGG-3ʹ and reverse 5ʹ-GAGAACGAGGACCCTGCAAT-3ʹ; GAPDH forward, 5′-GGTGATGCTGGTGCTGAGTATGT-3′and reverse, 5′-AAGAATGGGAGTTG -CTGTTGAAGTC-3′. All PCR reactions were repeated 3 times and negative control (C) was the non-template reactions. The 2-ΔΔCT method was used to normalize the fold change of gene expression levels to endogenous control GAPDH.