Rnalater reagent

To investigate molecular events associated with lingo-1 inhibition, changes in gene expression were evaluated by qPCR. The following primers were used for qPCR: Lingo-1, 5′-CTTTCCCCTTCGACATCAAGAC-3′ and 3′-CAGCAGCACCAGGCAGAA-5′; GAPDH, 5′-ACAGTCAGCCGCATCTTCTT-3′ and 3′-GACAAGCTTCCCGTTCTCAG-5′. GAPDH was used for normalization. qPCR was performed using unfixed ONs 28 days after ONC in the lingo-1-shRNA and NC-shRNA groups. ONs were dissected in ice-cold PBS and immediately immersed into RNAlater reagent (Qiagen GmbH, Hilden, Germany). RNA was extracted using a RNeasy kit (Qiagen GmbH) and reverse-transcribed using iScript cDNA Synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) to obtain cDNA. qPCR was performed using the iQ™ SYBR^® Green Supermix kit according to manufacturer's protocol (Bio-Rad Laboratories, Inc.). The following thermocycling conditions were used: Initial denaturation at 95°C for 10 min; 40 cycles of 95°C for 30 sec and 60°C for 1 min; and a final extension at 72°C for 1.5 min. The 2^−ΔΔCq method was used to quantify the relative changes in gene expression (32 (link)). The average C_q was calculated for the target gene and GAPDH and the ΔC_q (C_q,target-C_q,GAPDH) values were analyzed. All qPCR experiments were performed with three technical replicates.

GC tissue specimens and corresponding paracancerous gastric tissue specimens were obtained from 25 GC patients from Binzhou People's Hospital. Each patient was diagnosed with GC pathologically and none of the patients received any treatment before operation. The tissue samples were processed within 1 hour and submerged in RNAlater reagent from Qiagen GmbH (Hilden, Germany) for half an hour. After that, the samples were stored at -80 ˚C till RNA extraction. All tissue samples were examined and classified under the management of experienced pathologists.
As for blood samples, the present study totaled 134 participants, consisting of 94 patients with GC and 40 healthy controls. Postoperative blood specimens were also obtained from 25 patients with GC. All the blood samples were disposed within 2 h. Serum was disposed at 4 °C and recruited by centrifugation (1200 × g , 10 min). Another centrifugation (10000 × g, 10 mins, at 4 °C) was performed for completely removing residual cellular debris. The serum specimens were kept in liquid nitrogen till RNA extraction.
Our research was managed by the ethics committee of Binzhou People's Hospital. Consent statement was obtained from each participant. Table 1 lists the clinical characteristics of the GC patients.