Mastercycler realplex real time pcr system

Mouse peritoneal macrophages were cultured in 6-well plates (2 × 10⁶ cells/well) with RPMI 1640 (2 % FBS). The cells were incubated either with LPS (100 ng/ml) and SibaCec (0, 5, 10, and 20 μg/ml) dissolved in serum-free RPMI 1640 medium or incubated with SibaCec (10 μg/ml) alone. The cells incubated with serum-free RPMI 1640 and 100 ng/ml LPS were used as control. After treatment for 6 h, the cells were collected and total RNA was isolated. PrimeScript® Reverse Transcriptase (Takara, Japan) and SYBR green master mix (Takara, Japan) were used following the manufacturer’s instructions. qPCR was performed on a Realplex Mastercycler real-time PCR system (Eppendorf, Germany). The cycle counts of the target genes were normalized to the GAPDH gene, and accordingly the fold changes of the target genes were calculated. The primers used for qPCR are listed in Additional file 1: Table S1.

Total RNA was extracted from 10 silkworm antennae from 5 males per genotype using Trizol reagent (Invitrogen) and treated with RNase-free DNAse I (Ambion). cDNAs were synthesized using the Omniscript Reverse transcriptase kit (Qiagen) in a 20 μl reaction mixture containing 1 μg total RNA. Quantitative real-time RT-PCR (RT-qPCR) assays were performed using SYBR Green Realtime PCR Master Mix (Thermo Fisher Scientific) on an Eppendorf Real-time PCR System MasterCycler RealPlex instrument. RT-qPCR reactions were carried out with gene-specific primers (S1 Table). A 10-fold serial dilution of pooled cDNA was used as the template for standard curves. Quantitative mRNA measurements were performed in three independent biological replicates, and data were normalized to the amount of Bmrp49 mRNA [12 (link)].

Whole larvae samples were separately collected from silkworms treated with 10⁶ OBs/larva in the TG-1, TG-2, TG-3, and WT groups (n = 6 per group) at 72 hpi. The expression levels of several BmNPV genes (immediate early gene ie-1, early gene p143, late gene vp39, and very late gene p10) were analyzed by quantitative real-time RT-PCR (qRT-PCR). qRT-PCR assays were performed using SYBR Green Real-time PCR Master Mix (QPK-201C, Toyobo, Kusatsu, Japan) on an Eppendorf Real-time PCR System Mastercycler realplex. A 5-fold serial dilution of pooled cDNA was used as the template to make standard curves. Quantitative mRNA measurements were performed in three independent biological replicates and normalized to Bmrp49 mRNA.

Total RNA was extracted by TRIzol reagent (Invitrogen, CA, USA), and reverse transcription reactions were performed using the Superscript First-Strand cDNA synthesis system (Invitrogen, CA, USA). Real-time PCR was performed with Mastercycler-realplex Real-Time PCR System (Eppendorf, Germany) using a One-Step SYBR Prime Script TM RT-PCR Kit II (Takara, Tokyo, Japan) according to the manufacturer's instructions. The 2^-△△ct method was applied to calculate the levels of mRNA expression relative to that of GAPDH, and the values were presented by relative quantity.

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). Briefly, cells cultured on a 100-mm dish were lysed by applying 1 ml of Trizol reagent. Samples were segregated into phenol-chloroform phases. The aqueous supernatant phase was transferred to an RNase-free tube and precipitated with isopropanol. The RNA pellet was washed twice with 70% ethanol prepared with DEPC-treated water, air dried, and dissolved in RNase-free water (Thermo Fisher Scientific, Waltham, MA). The cDNAs were synthesized from the prepared total RNA using NCode miRNA First Strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacture's protocol. The amount of miRNA was detected with 5 Prime RealMasterMix SYBR ROX (5 Prime) and an Eppendorf Mastercycler realplex Real-Time PCR system. The quantitative real-time PCR runs were performed under the following thermocycler conditions: initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 55°C for 15 s, and 68°C for 20 s. Primers for mature miRNAs designed using their rat sequences from miRBase are listed in Table 1. The expression of miRNAs was normalized to 5S rRNA. 5S primers: forward 5′-GCCCGATCTCGTCTGATCT-3′; reverse 5′-GCCTACAGCACCCGGTATC-3′. U6 primers: forward 5′- GTGCCTGCTTCGGCAGCAC -3′; reverse 5′- GTGTCATCCTTGCGCAGGG-3′.

Total RNA, extracted using PureLink RNA Mini Kit (Ambion, Thermo Fisher Scientific, Milan, Italy) was used to evaluate NFκB p65, NLRP3, and IL-1β mRNA expression by real-time PCR. Then, one microgram of total RNA was reverse transcribed using M-MLV reverse transcriptase (M1302 Sigma-Aldrich) to synthesize cDNA for 10 min at 70 °C, 50 min at 37 °C, and 10 min at 90 °C. Three independent biological replicates were analyzed for each sample. Real-time PCR was performed with the Mastercycler real plex real-time PCR system (Eppendorf, Hamburg, Germany). Beta-2 microglobulin (B2M Hs.PT.58v.18759587, Tema Ricerca Srl) was used as an endogenous marker for data normalization. The expression levels of all mRNAs considered were evaluated in hGF cells cultured alone, in hGF cells cultured with Coldpac (Yates Motloid) and hGF cells cultured with ProTemp 4™ (3M ESPE ™) for 24 h and 1 week. Commercially available PrimeTime™ RELA (Hs.PT.58.22880470, Tema Ricerca Srl), NLRP3 (Hs.PT.58.39303321, Tema Ricerca Srl), IL-1β (Hs.PT.58.1518186) and PrimeTime™ Gene Expression Master Mix (cat. n°1055772, Tema Ricerca Srl) were used according to standard protocols (Table 1). Expression levels for each gene were obtained according to the 2^−ΔΔCt method ((relative quantification) [51 (link)]. Real-time PCR was performed in three independent experiments.