Total rna isolation kit

The total mRNA expression levels of NLRP3, Caspase-1, IL-1β, and IL-18 were analyzed via PCR; total RNA was extracted and purified using TRIzol (Invitrogen, CA, U.S.) and a Total RNA Isolation Kit (Takara, Otsu, Shiga, Japan). The purity of RNA was subsequently determined based on A260/A280 ratio. The mRNA was reverse-transcribed into cDNA using a Prime Script RT Reagent Kit (Takara) in accordance with manufacturer's instructions. Using SYBR Premix Ex Taq Kit (Takara, Japan) and specific primers, the expression levels of target gene mRNAs were measured with a Bio-Rad IQ5 Real-Time System (Bio-Rad, Hercules, CA). GAPDH was used as a housekeeping gene for mRNA analysis. The primer sequences are listed in Table 1.
The following thermal cycle condition was employed to reverse-transcribe the mRNA: an initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, and elongation at 72°C for 10 sec. Relative concentrations of target genes were determined based on the cycle threshold (Ct). The qRT-PCR data were exported and processed using the ΔΔCt method.

For quantitative reverse transcription PCR (qRT‒PCR) assays, total RNA was extracted from HNSCC cells using the Axygen Total RNA Isolation Kit and further reverse transcribed using the PrimeScript ™ RT Reagent Kit with gDNA Eraser (Takara, Japan). Then, synthesized cDNA was subjected to real-time PCR in the presence of a One Step SYBR^@ PrimeScript^@ PCR Kit (Takara, Japan) in an Applied Biosystems StepOne™ machine (Invitrogen, USA). The differential expression of target genes was evaluated and quantified using the formula 2^−ΔΔCT, with target gene expression levels normalized to that of the housekeeping gene GAPDH. The designed primer sequences used in the current research are listed in Supplementary Table 3.

Total RNA was extracted from homogenized cells using Total RNA Isolation Kit (Takara, Japan). The purity of obtained RNA was determined by A260/A280 ratio. 2.0 μg of RNA sample was reversely transcribed with PrimeScript RT Reagent Kit (Takara, Japan). The resulting cDNA was then amplified by using SYBR Premix Ex Taq Kit (Takara, Japan) with primer pairs specific to target genes as shown in Supplementary Table 1, which were finally normalized against the transcriptional level of housekeeping gene GAPDH. The thermal cycle condition was optimized as: initial denaturation at 95 °C for 30 s, denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, elongation at 72 °C for 10 s for 40 cycles. At the end of the reaction, a melting curve analysis (65–105 °C) was carried out to check for the presence of primer dimers. The relative concentration of target genes was determined by cycle threshold (Ct) at which specific fluorescence became detectable. The Ct value was used for kinetic analysis and was proportional to the initial number of target copies in the sample. The qRT-PCR data was exported and processed using the ΔΔCT method.

Total RNA was extracted with a Total RNA Isolation Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions and quantified using spectrophotometry (NANO drop 2000; Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences were: Col1a1, 5’-GAGGGCCAA GACGAAGACATC-3′ (sense) and 5’-CAGATCACGTCATCGCACAAC-3′ (anti-sense); MMP-1, 5’-AAAATTACACGCCAGATTTGCC-3′ (sense) and 5′-GGT GTGACATTACTCCAGA GTTG-3′ (anti-sense); MMP-2, 5’-GATACCCCTTTGA CGGTAAGGA-3′ (sense) and 5’-CCTTCTCCCAAGGTCCATAGC-3′ (anti-sense); MMP-9, 5’-AGACCTGGGCAGATTCCAAAC-3′ (sense) and 5’-CGGCAAGTCTT CCGAGTAGT-3′ (anti-sense); TIMP-1, 5’-AGAGTGTCTGCGGATACTTCC-3′ (sense) and 5’-CCAACAGTGTAGGTCTTGGTG-3′ (anti-sense); TIMP-2, 5’-GCTG CGAGTGCAAGATCAC-3′ (sense) and 5’-TGGTGCCCGTTGATGTTCTTC-3′ (anti-sense); TIMP-3, 5’-CATGTGCAGTACATCCATACGG-3′ (sense) and 5’-CATC ATAGACGCGACCTGTCA-3′ (anti-sense). The relative expression of mRNAs was evaluated by the 2^–ΔΔCt method and normalized to the expression of GAPDH. The cycling program involved preliminary denaturation at 95 °C for 1 min, followed by 45 cycles of denaturation at 95 °Cfor 10 s, annealing at 62 °Cfor 25 s, and elongation at 62 °C for 20 s, followed by a final elongation step at 70 °C for 5 min.

For genome annotation, we sequenced the transcriptomes of 500 adult females and males separately. Total RNA was extracted using the Takara RNIzol Total RNA Isolation Kit, following the manufacturer’s instructions. The cDNA libraries were constructed by NEBNext^® Ultra™ RNA library prep kit for Illumina^® (NEB, USA) and then sequenced on the Illumina NovaSeq 6000. The cDNA library construction and sequencing were performed by Novogene Co., Ltd. (Beijing, China).

Total RNA was isolated from silkworm (strain Dazhao) tissues using a total RNA Isolation Kit (TaKaRa, DaLian, China), followed by treatment with DNaseI to remove possible contamination from genomic DNA. cDNA was synthesized by PrimeScript™ Reverse Transcriptase (TaKaRa, DaLian, China), following the manufacturer's protocol. The cDNA was used as a template. The amplified products with gene-specific primers BmYki-1 and BmYki-2 were cloned into vector pMD19-T (TaKaRa, DaLian, China). BmYki cDNA was sequenced after the recombinant plasmids were identified.