Iq5 thermal cycler

Real time quantitative PCR (RT-qPCR) was performed to measure relative expression of the genes AQP and AGLU in B. tabaci. A total volume of 25μl reaction mixture contained 12.5μl SYBR^® Green Real-Time PCR Master Mix (Thermo Fisher Scientific, USA), 0.25μl each of the forward and reverse primers (Table 1;0.1 pmole), 2.5μl cDNA (~25 ng) and 9.5μl water. After optimization, the thermocycling conditions were: 1 cycle at 94°C for 10 min followed by 40 cycles, at 94°C (for 30 sec.), 57°C (for 30 sec.) and 72°C (for 30 sec.). The RT-qPCR was performed in a 96 well microtiter plate using Bio-Rad iQ5 thermal cycler (Bio-Rad, USA). Full experiments with all necessary controls were triplicated. At the end of every run, a melt curve analysis was performed from 60 to 95°C, with an increment of 0.5°C every 10 sec. in order to assess the specificity of the amplification product. Quantification results were analyzed by ΔΔCt method [37 (link)] and the 18S ribosomal RNA (rRNA) gene (GenBank accession number Z15051.1) was used to normalize the corresponding Ct values. Transcript levels were measured in whiteflies fed on transgenic plants and/or control plants and the fold change in the expression levels of AQP and AGLU were determined.

We selected 5 lncRNAs from the m6A-related network based on connectivity and 5 lncRNAs from 8 GO items enriched in ceRNA networks. First, a boxplot was used to reveal the expression levels of selected lncRNAs from four subgroups by the pheatmap and ggplot2 R packages. Then, the vegan package was used for the mantle order test, which is detailed in the following processes.
Total cellular RNA was extracted using a Quick-RNA Miniprep Kit (ZYMO Research, Irvine, CA, USA), and total RNA from mouse tissues was prepared with TRIzol (Life Technologies) according to the manufacturer’s instructions followed by reverse transcription and real-time quantitative polymerase chain reaction (qRT‒PCR) assays, as we described previously (L. Zhang et al., 2014). One microgram of total RNA was used for reverse transcription using M-MLV Reverse Transcriptase (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol and was detected using PowerUp SYBR Green Master Mix (Thermo Scientific) on a Bio-Rad iQ5 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). Differences in expression were evaluated by the comparative cycle threshold method using GAPDH or β-actin as a control. The primer sequences chosen for the qRT‒PCR experiments are listed in Table 2. Finally, Prism 8.0.2 was used to reveal the selected lncRNA expression levels by bar chart.

The RNA-seq data were verified by qPCR using the same RNA samples used for RNA-seq. The concentration of total RNA was measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). One μg of RNA was used as a template from which first-strand cDNA was synthesized using a SuperRT cDNA Synthesis Kit (CWBIO). QPCR was performed using an UltraSYBR Mixture Kit (CWBIO). The total volume of the qPCR mixture was 20 μL containing 10 μL of 2 × UltraSYBR Mixture (High ROX), 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 1 μL of cDNA, and 8.2 μL of ddH₂O. Reactions were carried out on a Bio-Rad iQ™5 Thermal Cycler (Bio-Rad) with the following reaction conditions: denaturation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 15 s, annealing and elongation for 1 min at 60 °C, and a melting curve process from 55 to 95 °C. Nine genes were selected as qPCR-verified targets. The primer sequences for these genes were listed in Additional file 8. Three biological replicates per groups and two technological replicates per sample were included in the qPCR. GAPDH was used as the endogenous reference gene, and the WSD2 group was set as the criterion. Expression levels of genes in each group were presented as expression folds relative to the criterion using the 2^-ΔΔCT method [67 (link)].

SIM A-9 cells were collected from plates and total RNA was extracted using a Quick-RNA Miniprep Kit (ZYMO Research, Irvine, CA, United States). 1 μg of total RNA was subjected to reverse transcription using M-MLV Reverse Transcriptase (Thermo Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. For qRT-PCR, PowerUp SYBR Green Master Mix (Thermo Scientific) was used, and experiments conducted on a Bio-Rad iQ5 thermal cycler (Bio-Rad Laboratories, Hercules, CA, United States). Differences in expression were evaluated by the comparative cycle threshold method using GAPDH as a control. To extract total RNA from mouse tissues, TriZol reagent (Life Technologies) was added to homogenized tissue according to the manufacturer’s instructions followed by qRT-PCR assays as we described previously (Qiu et al., 2020 (link)).