Dnase rnase free water

Mengovirus cDNA detection was used to validate the process: if a sample was negative for Mengovirus the analysis was repeated from the sample preparation step. Real-time PCR was performed using primers and TaqMan probe shown in Table 2 to confirm the process effectiveness.
The reaction was performed using RNA UltraSense™ One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA) in a total volume of 25 µL containing 5 µL of Ultrasense reaction mix (5×), 1 µL of each primer (12.5 µM and 22.5 µM, Forward and Reverse respectively), 1 µL of probe (6.25 µM), 0.5 µL of Rox reference dye (50×), 1.25 µL of RNA Ultrasense enzyme mix and 10.25 µL of DNAse-RNase-free water (Sigma–Aldrich, St. Louis, MO, USA). Five µL of RNA template were added to the reaction mix, and positivity was detected in each sample, highlighting data reliability.
The reaction was performed in a CFX96 Touch™ Real-Time PCR Detection System (Bio–Rad, Hercules, CA, USA). RT was performed for 1 h at 55 °C, and then samples were incubated at 95 °C for 5 min and amplified for 45 cycles of 15 s at 95 °C, 1 min at 60 °C and 1 min at 65 °C. Each analysis included a negative control which contained water in place of RNA.

The presence of DENV RNA was detected by RT-PCR using the PrimeScript™ One Step RT-PCR Kit Ver. 2 (Takara Bio Inc., Shiga, Japan) following the manufacturer’s protocol. Briefly, the RT-PCR amplification was carried out in a final volume of 25 μL with 5 μL of RNA. The RT-PCR mixture consisted of 1 μL of enzyme mix, 13 μL of 2× buffer, 4 μL of nuclease-free water, and 1 μL of 100 pmol forward and reverse primers, with separate primer sets (Supplementary Table S1) for the detection of DENV and the identification of specific DENV serotypes [27 (link),28 (link),29 (link)]. The RT-PCR program consisted of the following steps: 50 °C for 30 min; 94 °C for 2 min; 30 cycles at 94 °C for 30 s, 54 °C for 30 s, 72 °C for 1 min; and 72 °C for 7 min; and carried out using the SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA). DNase/RNase-free water (Sigma, New York, NY, USA) served as a negative control, while a mixture of all four DENV serotypes (DENV-1 (99St12Astrain), DENV-2 (00St22A), DENV-3 (SLMC 50 strain), and DENV-4 (SLMC 318 strain)) with a known viral concentrations (10⁶ FFU/mL of DENV-1, -3, and -4 and 10⁷ pfu/mL of DENV-2) was used as a positive control [30 (link)]. Amplified products were detected by agarose gel electrophoresis and visualized in a UV chamber to confirm the presence of DENV or determine the DENV serotype.

RNA was collected from cell lysates using the ZymoResearch MicroPrep kit (Genesee Scientific, San Diego, California). 10–100 ng of RNA was converted to cDNA using the qPCRBIO cDNA Synthesis kit from Genesee according to the manufacturer's guidelines for small amounts of RNA. The product of the cDNA reaction was diluted 1:3 with DNase/RNase-free water (Sigma Aldrich, St. Louis, Missouri, US) and applied to a 20 μl PCR reactions using the pre-designed primers from Sigma Aldrich (St. Louis, Missouri, US) that accompany the KicQ Start SYBR Green Master Mix (Sigma), using pre-determined annealing and amplification cycling, up to 40 cycles. Given the small quantity of RNA available applied to cDNA reactions each cDNA product was sufficient to run 5–6 gene targets in duplicate, in addition to the HK gene (GAPDH). PCR was performed on the ABI 7500 under fast cycling conditions, Ct values were determined and subsequent analysis was performed using the software StepOne Real-Time PCR System (ABI 7500). The delta delta Ct method was used to calculate fold-change of the various treatment condition to IL-2/IL-7 stimulated cells, or baseline.

For easy visualization and assessment of siRNA transfection efficiency, MISSION® siRNA Fluorescent Universal Negative Control #1, 6-FAM was used. According to the manufacturer's instructions, siRNA was dissolved in 1mL sterile DNAse/RNAse free water (Sigma-Aldrich). Cells were then transfected with 100 µL siRNA (25 and 50 nM) coupled with AuNPs-d-PLL-PEG-OCH₃ or AuNPs-d-PLL-PEG-FA (500 µg/mL) for four hours at room temperature. Cells were then washed and prepped for confocal microscopy.

Mengovirus detection was used to validate the process; samples with negative mengovirus amplification needed to be repeated from RNA extraction.
RT Real-time PCR was performed using primers and TaqMan probe shown in Table 2, to confirm the process effectiveness.
The reaction was performed using RNA UltraSense™ One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA) in a total volume of 20 μL containing 5 μL of Ultrasense reaction mix (5×), 1 μL of each primer (12.5 μM and 22.5 μM, Forward and Reverse, respectively), 1 μL of probe (6.25 μM), 0.5 μL of Rox reference dye (50×), 1.25 μL of RNA Ultrasense enzyme mix and 10.25 μL of DNAse-RNase-free water (Sigma–Aldrich, St. Louis, MO, USA). Five μL of RNA template were added to the reaction mix, and positivity was detected when Ct ≤ 40.
The reaction was performed in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Reverse transcription was performed for 1 h at 55 °C; samples were then incubated at 95 °C for 5 min and amplified for 45 cycles of 15 s at 95 °C, 1 min at 60 °C and 1 min at 65 °C. Negative and positive amplification controls were included in each run.