Sensiscript rt kit

RNA was extracted from sorted cells using an RNeasy RNA isolation kit (Qiagen) then reverse transcribed into cDNA with the Sensiscript RT kit (Qiagen). The cDNA was amplified for specific targets using TaqMan in a ViiA 7 Real-Time PCR System (Life Technologies). The thermal cycling parameters were 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were analysed with the 7500 Fast System SDS software. The TaqMan assays used are listed in Supplementary Table 1.

RNA was extracted using the NucleoSpin RNA XS kit (Macherey-Nagel, Düren, Germany) following manufacturer’s instructions yielding 100–250 ng total RNA per sample. Subsequent reverse transcription (Sensiscript RT kit, Qiagen, Hilden, Germany) and normalisation were conducted using 50 ng of cDNA. Out of six candidate genes, actin A1 (DV3000100) was chosen as a reference gene after optimisation tests [63 ] following the Normfinder procedure [64 (link)]. The primer pairs were designed based on the D. vitifoliae genome version 3.2 using the NCBI primer tool software (Table 1). Quantitative RT-PCR analyses were performed using the Rotor-Gene Cycler Q (Qiagen, Hilden, Germany) employing KAPA SYBR FAST qPCR Universal (Kapa Biosystems, Wilmington, US) as detector agent in technical duplicates (total reaction vol. 12 μl with 2 μl cDNA (1:7)). Primer efficiencies were determined by conducting standard curves with four step template dilutions. Cycling conditions were: one cycle for 5 min at 95°C, 45 cycles for 8 sec at 95°C, 20 sec at 60°C, 15 sec at 72°C, 3 sec at 75°C [48 (link)]. Relative gene expression levels were calculated using the EasyqpcR package (bioconductor) in R. Statistics were performed with Independent T tests (p < 0.05) in SPSS (IBM, v24) comparing the gene expression levels in salivary glands dissected from feeding larvae (SG0) versus starving larvae at 24 h (SG24).

Total RNA was isolated using RNeasy Mini Kit (Qiagen) from PBMCs (1 × 10⁶). Genomic DNA was removed from total RNA prior to cDNA synthesis using RNase-free DNase Set (Qiagen). First-strand cDNA synthesis was performed for each RNA sample using Sensiscript RT Kit (Qiagen). Random hexamers were used to prime cDNA synthesis.

Total RNA was isolated from cell pellets using RNeasy Mini Kit (Qiagen, Valencia, CA), and first-strand cDNA was subsequently synthesized using Sensiscript RT Kit (Qiagen) according to the manufacturer’s instructions. mRNA expression was determined by real-time PCR using SYBR Green Master Mix under standard thermocycler conditions (Applied Biosystems, Foster City, CA). Data were collected and quantitatively analyzed on an ABI Prism 7900 Sequence Detection System (Applied Biosystems). Mouse GAPDH gene was used as endogenous control for sample normalization. Results were presented as fold increases relative to the expression of mouse GAPDH. Sequences of PCR primers are listed in Table S2.

Single cell suspensions of isolated renal tissue from Sham-operated or CLP-treated mice were stained with 7-AAD to detect viable cells and an antibody cocktail containing CD326, CD45, F4/80, and CD11b (all purchased from BD). Cell suspensions were sorted using a FACS Aria (BD) FACS sorter, resulting in CD45-/CD326+ epithelial cells and CD45+/F4/80+/CD11b+ MΦ. The gating strategy is given in Supplementary Figure S1. Cells were then transferred to the cell culture and short-term cultured for 24 h using Dulbecco’s modified Eagle’s medium (Gibco, Dreieich, Germany) for TEC culture and RPMI-1640 medium for rMΦ, each supplemented with penicillin 100 U/mL (Sigma-Aldrich, Taufkirchen, Germany), streptomycin 100 mg/mL (Sigma-Aldrich), and 10% FCS (Capricorn Scientific, Ebersdorfergrund, Germany).
Supernatants were harvested for AAS and cellular lysates were used for RNA isolation. RNA isolation and transcription were performed using the RNeasy Micro Kit (Qiagen, 74004, Hilden, Germany) and Sensiscript RT Kit (Qiagen, 205211) according to the manufacturer’s kit protocols.