Nanovue plus spectrophotometer

Macrophage-like cells were directly lysed with 700 μl of QIAzol^® Lysis Reagent (Qiagen, Hilden, Germany). Total RNA extraction was performed with the miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. To quantify extracted RNA a NanoVue PlusTM spectrophotometer (GE Healthcare Life Sciences, Piscataway, NJ, United States) was used and the integrity/quality was assessed by 1% agarose gel stained with GelRed (Biotium, Hayward, CA, United States). For gene expression, 500 ng total RNA were reverse transcribed using PrimeScriptTM RT Master Mix (Perfect Real Time) (Takara Bio Inc.) according to the manufacturer’s instructions. The cDNA synthesis for three microRNAs (miR-126, miR-146a, and miR-346) was performed by TaqMan^TM MicroRNA Reverse Transcription Kit (Applied Biosystems) using 12.5 ng of total RNA.

Cells were directly lysed with 700 μL of QIAzol^® Lysis Reagent (Qiagen, Hilden, Germany). Total RNA extraction was performed with the miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The extracted RNA was quantified using a NanoVue Plus^TM spectrophotometer (GE Healthcare Life Sciences, Piscataway, NJ, USA). The cDNA synthesis was performed from 500 ng of total RNA using PrimeScript^TM RT Master Mix (Perfect Real Time) (Takara Bio Inc. Shiga, Japan) according to the manufacturer’s instructions.

To determine the release profiles of murine GM-CSF (80 ng/mL) and CpG ODN1826 (45 μg/mL) from the peptide hydrogel, each agent was incorporated into hydrogel as described above. The total volume of the mixture was 100 μL. Triplicate samples were plated in each well of a 12-well cell culture plate. After the gelation was confirmed, PBS was added into each well followed by placement into a 37 ^°C CO₂ incubator. Supernatants were collected and the same volume of fresh PBS was replenished at each time point (0, 0.5, 1, 3, 6, 12, 18, 24, and 48 h). The in vitro release kinetics of GM-CSF or CpG from the hydrogel was measured using a murine GM-CSF ELISA kit (eBioscience, Inc., San Diego, CA, USA) or NanoVue Plus^TM Spectrophotometer (GE Healthcare Life Sciences, Pittsburgh, PA, USA), respectively.

Total RNA was extracted from PBMCs and frozen glioma samples using TRIzol reagent (Invitrogen, France) as previously described (24 (link)). RNA concentration and quality were measured using the NanoVueTM Plus Spectrophotometer (GE Healthcare, UK), then cDNA was synthesized using Tetro Reverse Transcriptase Enzyme (Bioline, France) from 0.5 μg of total RNA in a 20 μl of reaction mixture according to the manufacturer’s instructions, mixed with 1 μl of Random Hexamer Primer 25µg (Bioline, France) and 4 μl of RNase-Free water, then incubated at 70°C for 5 min to break the secondary structures of RNA.
Next, 4 μl of Tetro Reverse Transcriptase buffer, 4 μl of dNTP (10 mM), 0.5 μl of RNase Inhibitor (Invitrogen, France), 0.5 μl of Tetro Reverse Transcriptase Enzyme (Bioline, France), and 1 μl of RNase-Free water were added and incubated at 25°C for 10 min then at 45°C for 30 min then at 85°C for 5 min.

Total RNA from 60 fresh biopsies was isolated using Trizol reagent (Invitrogen, France) (42 (link), 43 (link)). We analyzed RNA concentration and purity with the use of a NanoVueTM Plus Spectrophotometer (GE Healthcare, UK). The samples were then diluted with ultrapure water to ensure that each tube had the same concentration of RNA. According to the manufacturer’s instructions, cDNA was synthesized from 1 μg of RNA included in a 20 μl reaction mixture containing RNase-Free Water Random Hexamer Primer (Bioline, France) and incubated at 70°C for 5 min. Afterward, 1 µL RNase-free water, 4 µL Tetro reverse transcriptase buffer, 0.5 µL RNase inhibitor (Invitrogen, France), 4 µL dNTP (10 mM), and 0.5 µL Tetro reverse transcriptase enzyme (Bioline, France) were added, followed by incubation at 25°C for 10 min, then at 45°C for 30 min, and finally at 85°C for 5 min.

The papaya leaves (50–100 mg) were homogenized with liquid nitrogen. Total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s protocol. The yield and the quality of RNA were analyzed using a NanoVue^TM Plus Spectrophotometer (GE Healthcare, Freiburg, Germany).

Tissue samples were collected in RNA later solution (Ambion, United States) and homogenized using a hand-held tissue grinder (G-Biosciences, United States). DNA and RNA were extracted using Allprep DNA/RNA/protein kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA was reverse transcribed to cDNA using High capacity cDNA reverse transcription kit (Invitrogen, Applied Biosystems, Foster City, United States) according to the manufacturer's instructions. The quantity and quality of the isolated genomic DNA and cDNA were confirmed by Nano-Vue^TM plus spectrophotometer (GE Healthcare, Little Chalfont, United Kingdom) and agarose gel electrophoresis respectively. To assess the quality of extracted DNA and cDNA, PCR was carried out for the human β-globin gene using standard PCO3 and PCO4 oligonucleotides as described previously (17 (link)). Five samples that were negative for human ß-globulin gene amplification were excluded from the study and all other 80 samples were subjected to HLA-G isoform expression, HPV association, and gene expression studies.