Rnase r epicentre

Sample labeling and array hybridization were performed based on the manufacturer's protocol (Arraystar, Inc.). Total RNA was digested with RNase R (Epicentre; Illumina, Inc.) to remove linear RNA. The amplified circRNAs were then transcribed into fluorescent complementary RNA (cRNA) using a random priming method (Arraystar Super RNA Labeling kit; Arraystar, Inc.). The labeled cRNA were purified using the RNeasy Mini kit (Qiagen, Inc.) according to the manufacturer's protocol. The concentration of the labeled cRNA (pmol Cy3/µg cRNA) was measured using NanoDrop-1000 (Thermo Fisher Scientific, Inc.). For each sample, the labeled cRNA was processed as per a previously published protocol (27 (link)).

According to the Log2FC absolute value, the top five upregulated and downregulated DEcircRNAs were selected as candidate DEcircRNAs for further investigation. RT-qPCR was performed to determine the relative expression levels of 10 candidate circRNAs in tumor and paired Ctrl tissues derived from 60 patients with TSCC. Briefly, total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The detection of circRNAs was performed following digestion of the linear RNA using RNase R (Epicentre; Illumina, Inc.). Subsequently, the synthesis of cDNA was carried out (denaturation at 65°C for 5 min, reverse transcription at 42°C for 18 min and inactivation at 98°C for 5 min) using the PrimeScript™ RT reagent kit (Takara Bio, Inc.), followed by its amplification using SYBR-Green Premix DimerEraser™ (Takara Bio, Inc.). Lastly, the relative expression levels of the circRNAs were calculated using the 2-ΔΔCq method (16 (link)). The thermocycling conditions for qPCR were as follows: Pre-denaturation at 95°C for 3 min; followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 61°C for 20 sec. GAPDH was used as an internal reference for circRNAs. The primers are shown in Table SI.

Sample labeling and array hybridization were performed according to the manufacturer's protocol (Arraystar human circular RNA v2; design ID 074301). In brief, total RNAs were digested with RNase R (Epicentre; Illumina, Inc.) to remove linear RNAs and enrich circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent complementary (c)RNAs using a random priming method according to the Arraystar's protocol (Super RNA Labeling Kit; Arraystar, Inc.). The labeled cRNAs were purified using an RNeasy Mini Kit (Qiagen GmbH). The concentration and specific activity of the labeled cRNAs (pmol Cy3/µg cRNA) were measured using a NanoDrop ND-1000. Thereafter, 1 µg of each labeled cRNA was fragmented by adding 5 µl of 10X blocking agent and 1 µl of 25X fragmentation buffer, followed by heating at 60˚C for 30 min. Subsequently, 25 µl of 2X hybridization buffer was added to dilute the labeled cRNA. Next, 50 µl of hybridization solution was dispensed into the gasket slide and assembled on the circRNA expression microarray slide. The slides were incubated for 17 h at 65˚C in an Agilent Hybridization Oven (Agilent Technologies, Inc.). The hybridized arrays were washed, fixed and scanned using an Agilent Scanner (G2505C; Agilent Technologies, Inc.).

Briefly, total RNA was digested with RNase R (Epicentre; Illumina, Inc., San Diego, CA, USA) to remove linear RNAs and enrich for circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling kit; Arraystar, Inc., Rockville, MD, USA). The labeled cRNAs were purified by RNeasy Mini kit (Qiagen GmbH, Hilden, Germany). The concentration and specific activity of the labeled cRNAs (pmol Cy3/µg cRNA) were measured by NanoDrop ND-1000. Each labeled cRNA (1 µg) was fragmented by adding 5 µl 10X Blocking Agent and 1 µl of 25X fragmentation buffer, and then heated the mixture at 60°C for 30 min. Finally, 25 µl 2X Hybridization buffer was added to dilute the labeled cRNA. 50 µl of hybridization solution was dispensed into the gasket slide and assembled to the circRNA expression microarray slide. The labeled cRNAs were hybridized into an Arraystar Human circRNA array (8×15K; Arraystar, Inc.) and incubated for 17 h at 65°C in an Agilent Hybridization Oven (Agilent Technologies, Inc., Santa Clara, CA, USA). After the slides were washed, the arrays were scanned using an Agilent Scanner G2505C.

The stability of circHIPK3 was determined by RNase R. In short, 2 mg of total RNA was incubated at 37 °C for 30 minutes with or without 5 U/µg RNaseR (Epicentre; Illumina Inc), and then purified with the RNase MinElute cleaning kit (Jianlun Biotechnology Co., Ltd. Guangzhou, China) and analyzed with QRT-PCR.

RNA from three pairs of selected samples was subjected to microarray analysis according to the manufacturers protocol (Arraystar, Inc.). Briefly, total RNA was digested with RNase R (Epicentre; Illumina, Inc., Madison, WI, USA) to remove linear RNAs and enrich for circRNAs. Subsequently, the enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method (Arraystar Super RNA Labeling kit; Arraystar, Inc.). The Cy3-labeled cRNAs were purified using the RNeasy Mini kit (Qiagen GmbH). Each labeled cRNA (1 µg) was fragmented by adding 5 µl 10X blocking agent and 1 µl 25X fragmentation buffer, after which the mixture was incubated at 60°C for 30 min and was then diluted in 25 µl 2X hybridization buffer. A total of 50 µl hybridization solution was dispensed into the gasket slide, which was assembled onto the circRNA expression microarray slide. The slides were incubated for 17 h at 65°C in a hybridization oven (Agilent Technologies, Inc., Santa Clara, CA, USA). The hybridized arrays were washed, fixed and scanned using an Agilent Microarray Scanner system (catalog no. G2505C; Agilent Technologies, Inc.).