M mlv reverse transcriptase reagent

Total RNA was purified from 10 flies per each genotype (five males and five females) using Trizol Reagent, according to the manufacturer’s instructions (Thermo Fisher Scientific). cDNA was prepared from DNase I-treated RNA samples using the M-MLV Reverse Transcriptase reagent (Promega) and random hexamers. Diluted cDNA samples were quantitatively analyzed by SYBR Green-based Prime Q-Mastermix (GeNet Bio) and gene-specific primers using the LightCycler 480 real-time PCR system (Roche). To validate the efficiency of transgenic RNA interference, total RNAs from head or body extracts were analyzed similarly.

Total RNA was extracted from the cells using TRIzol (Invitrogen), and the purity of the RNA was assessed spectrophotometrically (A260/A280>1.8). Approximately 1μg of total RNA was reverse transcribed into cDNA using an MMLV reverse transcriptase reagent (Promega, Madison, WI, USA). According to the manufacturer’s instructions, real-time qRT-PCR was used to detect LINC00467 and NF-kb-p65 mRNA expression using a SYBR Green PCR Master Mix on an ABI 7500 sequence detection system (Life Technologies). All experiments were performed in triplicate using β-actin or U1 as internal controls. Relative expression levels were calculated using the 2^-ΔΔCt method. The following primer sequences were used: LINC00467-F: 5-TCGTCTTCAGGAAGCCAGAC-3; R: 5- TGGAAATCAAAAGGGTCAGC-3; NF-kb-p65 -F: 5-ATGTGGAGATCATTGAGCAGC-3; R: 5-CCTGGTCCTGTGTAGCCATT-3; β-actin -F: 5-CATGTACGTTGCTA TCCAGGC-3; R: 5-CTCCTTAATGTCACGCACGAT-3; U1 -F: 5- GGGAGATACCATGATCACGAAGGT-3; R: 5- CCACAAATTATGCAGTCGAGTTTCCC-3.

The RNAs were extracted via an Rneasy Mini kit (Qiagen, Inc., Valencia, CA, USA). Then, reverse transcription experiments were conducted on the RNAs through the usage of miRNA cDNA Synthesis reagent (Vazyme, Nanjing, China) or M-MLV Reverse Transcriptase reagent (Promega, Madison, WI, USA) in line with the protocols. Next, qRT-PCR was manipulated by using SYBR Green qPCR mix (Takara, Dalian, China). The 2^−ΔΔCt method was employed to compute the expression. GAPDH and U6 served as internal controls. The primers used are exhibited in Table 1.