Chamq sybr colour qpcr master mix

Total RNAs from the heads of honey bee from four typical and four single-cohort colonies were used for quantitative PCR analysis. Briefly, the first strand cDNA was obtained using a HiScript II Q RT SuperMix for qPCR (Vazyme, China) and were subjected to quantification of lncRNAs or mRNAs with β-actin as the housekeeping gene using a standard SYBR Green PCR kit (ChamQ™ SYBR Colour Qpcr Master Mix) (Vazyme, China) on the CFX Connect Real-Time System (Bio-Rad). The quantitative PCR was performed under the following conditions: 95 °C for 30 s, 40 cycles of 95.0 °C for 10 s and 60 °C for 30 s. Then, for melting curve analysis, temperatures were increased from 70 °C to 95 °C (at 0.5 °C increment every 5 s until plate reading). Each sample test was performed in triplicate for all reactions. Gene expression was quantified relative to the expression of β-actin using the comparative cycle threshold (ΔCT) method.

Res (99%) was obtained from Aladdin (Shanghai, China). Polyoxyethylene (40) stearate (Myrj 52) and dimethyl sulfoxide were purchased from Sigma-Aldrich (St Louis, MO, USA). Stearic acid and chloroform were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Lecithin, ethylene diamine tetraacetic acid (EDTA), bovine serum albumin (BSA), 4′,6-diamidino-2-phenylindole (DAPI) and 1% Alizarin Red S (ARS) solution at pH 4.1 were obtained from Solarbio Co. (Beijing, China). Cell counting kit-8 (CCK-8) and 4% paraformaldehyde were purchased from Biosharp (Shanghai, China). An osteogenic induction medium was obtained from Cyagen (Guangzhou, China). The alkaline phosphatase (ALP) staining kit was obtained from Yeasen (Shanghai, China). Osteocalcin (OCN) primary antibody, CD31 primary antibody and Cy3-labelled secondary antibody were obtained from Affinity Biosciences (Jiangsu, China). HiScript II QRT SuperMix for quantitative PCR (qPCR) and ChamQ SYBR Colour qPCR Master Mix were obtained from Vazyme (Nanjing, China). Dulbecco’s Modified Eagle’s Medium/Hams F12 (DMEM/F12, 1:1), foetal bovine serum and penicillin-streptomycin were obtained from Gibco (Grand Island, NY, USA). The GelMA hydrogel was obtained from EFL (Suzhou, China). All other chemicals were of analytical grade.

Total RNA was extracted with a Plant RNA pure kit (ZOMANBIO, ZP405‐2), then the reverse transcription of the cDNA was synthetized with HiScript III RT SuperMix for qPCR (Vazyme). The primers for amplification of all genes were designed by Primer Premier v. 5 software. The qPCR was performed by a LightCycler 96 Real‐Time PCR detection system (Roche, http://technical‐support.roche.com) using ChamQ SYBR Colour qPCR Master Mix (Vazyme, Q411‐02/03). The relative expression levels of genes were calculated by the 2^−ΔΔCt method and all reactions were repeated three times. Three independent biological replicates were used. The sequences of all primers are shown in Table S8.