Cdna reverse transcription kit

cDNA was prepared from each RNA sample using a cDNA Reverse transcription kit (Biorad). Each cDNA (20 ng) was subjected to qRT-PCR using Applied Biosystems TaqMan assays for selected genes on a One Step Plus instrument (Applied Biosystems). The number of cycles (Ct) needed to reach the midpoint of the linear phase was noted and all observations were normalized against the housekeeping gene GAPDH.

The cDNAs of DENV genome were synthesized using a cDNA reverse transcription kit (170-8890; Bio-Rad) and quantified by qPCR with specific probes. The expression of mosGCTL genes was measured by qPCR with SYBR supermix (170-8880; Bio-Rad). The primers and probes were shown in Table S3. Gene quantities were normalized using A. aegypti actin (AAEL011197).

RNAs were isolated with Trizol (Invitrogen Inc.) from cells or snap-frozen tissues. Reverse transcription was performed with the cDNA Reverse Transcription Kit (Bio-Rad) with iQ SYBR Green Supermix (170-8880). qRT–PCR was carried out using the ABI Prism 7900 Sequence Detector System (Perkin-Elmer Applied Biosystems) using Gapdh as an internal control. Each analysis was performed in triplicates and the results were normalized to Gapdh for each sample. The qRT–PCR primer sequences are listed in Supplementary Table 1.

RT-PCR was performed as previously described (Chen et al., 2014 (link)). Total RNA was isolated using TRIZOL reagent according to the manufacturer’s protocol. The cDNA was synthesized using a commercially available cDNA reverse transcription kit in a Bio-Rad thermocycler. Polymerase chain reaction (PCR) amplification was performed using the following cycle: an initial denaturing at 94°C for 5 min, followed by 30 cycles of denaturing at 94°C for 30 s, annealing at 58°C (GAPDH) for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The detailed information of specific primers is shown in Table 1. The relative mRNA level was calculated by normalization to GAPDH.

The Trizol reagent (Invitrogen, United States) was used for RNA extraction. The reverse transcription was then performed with the cDNA Reverse Transcription Kit (Bio-Rad, United States). Lentiviral vectors pCDH-CMV-MCS-EF1-copGFP-T2A-Puro-BDNF and pCDH-CMV-MCS-EF1-copGFP-T2A-Puro-GDNF vectors were constructed using PCR. The primer sequences were as follows: BDNF, 5′-GCG GGA TCC GCC ACC ATG GTG ACC ATC CTT TTC CTT AC-3′ and 5′-GCG GCG GCC GCC TAT CTT CCC CTT TTA ATG G-3′; GDNF, 5′-GCG GGA TCC GCC ATT ATG GGA TGT CGT GGC TG-3′ and 5′-GCG GCG GCC GCT CAG ATA CAT CCA CAC CGT TTA GC-3′. The lentiviral packaging vectors (pLP1, pLP2, pLP) were co-transfected along with pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, pCDH-CMV-MCS-EF1-copGFP-T2A-Puro-BDNF, or pCDH-CMV-MCS-EF1-copGFP-T2A-Puro-GDNF into 293T cells using Lipofectamine 2000 (Invitrogen, United States). After 48 h of transfection, the lentiviruses were collected after filtering the supernatant of cell culture medium.

RNAs were isolated with Trizol (Invitrogen Inc.) from snap-frozen tissues. Reverse transcription was performed with the cDNA Reverse Transcription Kit (Bio-Rad) with iQ SYBR Green Supermix (170–8880). qRT–PCR was carried out using the Bio-Rad CFX96 Real-Time System using GAPDH as an internal control. Each analysis was performed in triplicates and the results were normalized to GAPDH for each sample. The qRT–PCR primer sequences are listed in Supplementary Table 1. Relative expression was calculated using Comparative Ct method^{66 (link)}.