Rna blood mini kit

The research involved peripheral blood mononuclear cell (PBMC) gene expression analysis of CD4, CD8, CTLA4, GZMB, FOXP3, IL10, IL4, ILR2A, NOTCH, PDCD1, PRF1, TNFRSF18, TGFB, and TNFA genes, referenced to 18S rRNA. 2 × 10⁶ PBMCs were isolated from heparinized blood using density gradient centrifugation on Histopaque 1.077 (Sigma) and washed with PBS. The RNA was purified with RNA Blood Mini Kit (Qiagen) including genomic DNA removal with RNase-free DNase (Qiagen), according to the manufacturer's protocol. The samples were reversely transcribed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). 10 μl of final reaction volume in 100 μl of TaqMan PCR Master Mix was applied to each channel of a custom-designed low-density array (TaqMan) and analyzed on a TaqMan 7900HT instrument. The results are presented as ΔCT = CT_gene − CT_18S or as ΔΔCT = ΔCT_{mean control sample} − ΔCT_{test sample}. The mean control sample was the mean value of ΔCTs in the control group.

Total RNA was extracted from PBMCs and biopsies using RNA Blood Mini Kit (Qiagen) and RNeasy (Qiagen), respectively. HIV-1 RNA was detected by real-time PCR using the Generic HIV^® assays (Biocentric), according to manufacturer’s instructions.

LDH serum level and NLR were assessed at baseline; NLR was recorded after 3 months of treatment with anti-PD-1 ICI. Blood samples from enrolled patients were collected at baseline to conduct a gene profile analysis. RNA from PBMCs was extracted using RNA blood mini-Kit (Qiagen). Purified RNA was used for hybridization and underwent to gene profiling analysis on NanoString nCounter through PanCancer IO 360™ panel, characterized by human genes associated with immune activation, inflammation and control of the cell cycle. Gene data were normalized using nSolver Version 4.0 Software; NanoString. Counts were normalized to External RNA Controls Consortium (ERCC) technical controls and 30 housekeeping genes.

Ribonucleic Acid (RNA)was extracted from cell lysates using the RNA Blood Mini Kit and RNeasy^® Mini Kit (QIAGEN) according to the manufacturer’s instructions. The RNA was reverse transcribed using the ReverTra Ace qPCR RT Kit and ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). The relative FC in expression of the target normalized to expression of the corresponding control was calculated by the comparative Ct method. Primers are described in Table 1.

Approximately 30mg of tissue were homogenized (OMNI-TH homogenizer with dispersible tips) and total RNA was extracted thereof using the RNA Blood Mini Kit from Qiagen (Hilden, Germany) according to the manufacturer's instructions. DNase treatment was included. RNA concentration and quality were determined by spectrophotometry (NanoDrop ND-1000) and gel electrophoresis. One microgram of total RNA was reverse transcribed in a 20μl reaction comprising 20pmol CDS-primer (5′-T_n=30VN-3′), 100nM of each dNTP, 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl₂ 10mM DTT, 20U RNasin und 200U SuperScriptII (GibcoBRL). To allow optimal annealing, primer and RNA were incubated at 70°C for 10 min and cooled down on ice. The remaining reagents were then added in form of a master mix and cDNA synthesis was done at 42°C for one hour. The reaction was stopped at 70°C for 15 minutes and stored at −80°C until PCR analyses.

Twelve female C57BL/6JRj were randomly divided into four groups. Two groups (tetracycline-positive groups) were pre-treated for 48 h with 1 mg/mL of doxycycline and 5% sucrose in the drinking water. Doxycycline exposure was maintained throughout the entire experiment and fresh doxycycline solutions were prepared every 2–3 days. The tetracycline-negative groups received water with 5% sucrose. On day 0, mice were infected intraperitoneally with 5000 parasites. Each group was infected with a different strain: NY-SM + (pre-induced with tetracycline in vitro), NY-SM—(not induced with tetracycline), ADKIN A + (NY-SM cells transfected with RNAi insert Tb927.6.2300 and pre-induced with tetracycline in vitro), and ADKIN A—(NY-SM cells transfected with RNAi insert Tb927.6.2300 and not induced with tetracycline). Parasitaemia and anaemia were determined using a Neubauer improved haemocytometer. To confirm adequate knock-down of the genes of interest, 100 µL of blood was collected at day 4 post-infection (dpi). RNA was extracted using the RNA blood mini kit (QIAGEN) and subjected to RT-qPCR to determine the percentage of gene knock-down.
The role of 4EIP RNAi-mediated knockdown (NY-SM cells transfected with RNAi insert Tb927.9.11050) and knockout (4EIP-KO and 4EIP-AB strains) in in vivo infectivity was evaluated in a similar manner as described above.