Abi 7500 cycler

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. A cDNA Reverse Transcription kit (Takara, Tokyo, Japan) was used to synthesize cDNA from 2 µg of total RNA. The SYBR Green Master Mix (Roche, Basel, Switzerland) was used for qRT‐PCR on an ABI 7500 cycler (Applied Biosystems, Foster City, CA, USA). Thermal settings were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. The mean of the housekeeping gene β‐actin was acted as an internal reference for mRNA and small nuclear RNA U6 as the internal control for micro miRNA. qRT‐PCR was performed three times. The primers used in the experiment are listed in Table S3, and the results were analysed using the 2^−ΔΔCt relative expression method.

Trizol reagent (Invitrogen) was used to extract RNA. Total RNA was reverse-transcribed and subjected to Taqman miRNA Assay (Applied Biosystems, Foster City USA) following the manufacturer’s protocol. Sequences of miRNA-specific primers and probes (U6 served as an internal control) are identical as described before [4 (link)]. Data are shown as fold-change according the 2^ΔΔCT method. Reactions were performed using an ABI7500 cycler (Applied Biosystems, Foster City, USA). Fold changes of gene expression are displayed as results.

Total RNA extraction and quantitative real time PCR (qRT-PCR) were carried out by previously described methods [32 (link)]. First-strand cDNA was reverse transcribed from DNase I-treated RNA with oligo (dT) as the primer. qRT-PCR experiments were performed with the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) in a ABI7500 cycler (Applied Biosystems) with three technical repeats for each sample. Reactions were initiated at 94 °C for 15 min followed by 40 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s. The relative quantitation of the target gene expression level was performed using the comparative Ct (threshold cycle) method. The amplification of the PP2AA3 gene (encoding protein phosphatase 2A subunit A3, At1g13320) was used for an internal control [33 (link)]. Primers used for qRT-PCR are listed in Table S2.

Tissue specimens were collected from 48 control and 21 HNSCC patients from the Department of Pathology, First Affiliated Hospital of Guangxi Medical University (Nanning, Guangxi Medical University). Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissues using an FFPE RNA kit (Omega Bio-Tek, Norcross, GA, USA). RNA was reverse-transcribed into cDNA using the Mir-X miRNA qRT-PCR SYBR Kit (Takara, California, USA). The cDNA samples were processed for qPCR with SYBR-Green Master Mix (Takara, Tokyo, Japan) on an ABI 7500 cycler (Applied Biosystems) under the following conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 34 s. The expression of miR-221-3p in HNSCC tissues relative to negative control (NC) tissues was calculated using the 2- ΔΔCT method, with U6 as the internal control. The primers for miR-221-3p and U6 were synthesized by TaKaRa (Dalian, Liaoning, China), and the sequences were as follows: miR-221-3p: forward 5′-AGCUACAUUGUCUGCUGGGUUUC − 3′ and reverse 5′ mRQ 3′; U6: forward 5′-GGAACGATACAGAGAAGATTAGC-3′ and reverse 5′-TGGAACGCTTCACGAATTTGCG-3′. All the experiments were repeated three times.

Total RNA was extracted from cocultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. The RNA concentration and the A260/A280 ratio were determined using a UV spectrophotometer.
Primers were designed with Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/), and the sequences were checked by performing a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Table 1). In general, 2 μg total RNA was reverse transcribed in a 20 μl reaction using the following programs: 37°C for 15 min and 85°C for 5 s using PrimeScript RT kit (DRR047A, Takara, Japan). Then, 1 μl of cDNA was used for real-time PCR analysis in a 13 μl reaction volume using Premix Ex Taq II RT-PCR kit (DRR081A; Takara). Amplification efficiency differences between target genes and housekeeping genes were identified by comparing the slopes of the standard curves. The PCR reactions were run on an ABI 7500 cycler (Applied Biosystems) using the following programs: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 34 s. Analyses were performed using the 7500 system software (Applied Biosystems).

Total RNA was extracted from different samples (100 mg) using the Trizol reagent (Invitrogen) and following the manufacturer’s instructions. The first-strand cDNA synthesis was reverse transcribed from DNase I-treated RNA with oligo (dT)18 as the primer. qRT-PCR reactions were performed with the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) in a ABI7500 cycler (Applied Biosystems) with three technical repeats for each sample. Reactions were initiated at 94 °C for 15 min followed by 40 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The relative quantitation of the target gene expression level was performed using the comparative Ct (threshold cycle) method. The amplification of the PP2AA3 gene (encoding protein phosphatase 2A subunit A3, At1g13320) was used for an internal control. Primers used for qRT-PCR are listed in Table S1.