Rneasy plus mini kit 250

RNA was extracted from FACS-sorted monocytes using a RNeasy Plus Mini Kit (250) (Qiagen, Chatsworth, CA, USA) and reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen). PCR was performed on a Chromo4 real-time PCR system (Bio-Rad, Hercules, CA, USA) with GoTaq Green Master Mix (Promega, Woods Hollow, WI, USA), as described previously [10 (link)–12 (link)]. The mRNA expression of each gene was normalized to the expression of the reference gene β-actin. The specificity of PCR amplification was confirmed by melting curve analysis and agarose gel electrophoresis.

Mosquitoes treated as described above were collected, and DENV-2 RNA copies in each mosquito were detected by real-time RT-PCR at the same time, and the cycle threshold (C_T) value of each mosquito was recorded. At least 60 mosquitoes were analyzed for each group.
After being frozen to death at −20°C, each mosquito was collected in a grinding tube with 350 μl lysis buffer and then was fully grinded by tissue grinded instrument. The DENV-2 RNA was isolated with RNeasy plus Mini Kit (250) (Qiagen, German), and quantitatively tested with the dengue virus 2 real-time fluorescent RT-PCR kit (Shanghai ZJ Bio-Tech, China). The Master Mix volume for each reaction was pipetted as follows: super mix 18 μl, enzyme mix 1 μl, internal control 1 μl, extraction RNA 5 μl. PCR reaction conditions were: one cycle of 45°C for 10 minutes and 95°C for 15 minutes, then 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds, fluorescence measured at 60°C. During the bioassay, the standard curve between C_T values and DENV-2 RNA concentrations (copies/ml) was also detected as described previously [47 (link)].