Sybr green pcr master mix kit

TRIzol reagent was used to extract total RNA from cells and LAD tissues (Invitrogen, Waltham, MA, USA). Using a Reverse Transcription Kit, an equivalent quantity of RNA was reverse transcribed into cDNA (Promega, Madison, WI, USA). The ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used to run RT-PCR using the SYBR-Green PCR Master Mix kit (Promega, Madison, WI, USA). The gene expression was measured using the 2^–ΔΔCt of each response. The internal control was GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The primer sequences for qRT-PCR are listed in Table 1.

Total 380 second-instar nymphs of R. dorsali were microinjected with dsDCR2 or dsGFP (0.1 μg/μl) using Nanoject II Auto-Nanoliter Injector (Spring, USA) as described previously47 (link), then fed on diseased plants for 1 day. At different days padp, ten live and total dead leafhoppers were individually sampled daily for 18 days. Total RNA was extracted from individual leafhoppers with Trizol reagent (Invitrogen, USA). The cDNAs were synthesized using gene-specific primers of the major outer capsid protein P8 gene of RGDV in Table S2. RT-qPCR was performed using the SYBR Green PCR MasterMix kit (Promega, USA) as described above. Cycle thresholds (CT) were obtained by the RT-qPCR assay. The viral genome copy was calculated as the log of the copy number per microgram of insect RNA by mapping the CT value to the standard curve of the major outer capsid protein P8 gene of RGDV (y = −3.06x + 54.44). In addition, the relative abundance of DCR2 gene was confirmed by RT-qPCR as described already. Furthermore, midguts and salivary glands of viruliferous leafhoppers treated with dsGFP or dsDCR2 (n = 100) were dissected, immunolabelled with viral particle-specific IgG conjugated to rhodamine (virus-rhodamine) and the actin dye phalloidin-FITC (Invitrogen, UAS), and detected by immunofluorescence microscopy at 5 or 10 days pads as described previously26 (link).

To obtain the sequences encoding the core components of the RNAi pathway in the transcriptome of R. dorsalis, we used a local BLASTN search with sequences of N. lugens (JX023532.1, JX644040.1, KC316038.1 and JX644040.1 for DCR2, DCR1, AGO2 and AGO1, respectively) as queries45 (link). The obtained gene sequences for DCR2, DCR1, AGO2 and AGO1 of R. dorsalis were deposited in GenBank with accession numbers KT191018, KT191019, KT191020 and KT191021, respectively.
VCMs were inoculated with RGDV at a MOI of 10 for 2 h as described previously43 (link)44 (link). Total RNA was extracted with Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions at 72 hpi. The relative transcript abundance of RNAi pathway genes in RGDV-infected or mock-infected VCMs was estimated using RT-qPCR with the SYBR Green PCR MasterMix kit (Promega, USA) with the primers in Table S2, as described previously46 (link). The relative abundance of the RNAi pathway genes was normalized to an internal control gene actin and estimated by the 2^−ΔΔCt (cycle threshold) method.

At 14 days padp, total RNAs or proteins were extracted from the salivary glands of 30 viruliferous or nonviruliferous leafhoppers for to detect the relative transcript or protein levels of Rab27a and Rab5 during viral infection. RT-qPCR was performed using the SYBR Green PCR Master Mix kit (Promega, Madison, WI, USA). The actin transcript of N. cincticeps served as the internal reference, and the relative gene expression levels were calculated using the 2^-ΔΔCT method. For western blot assay, RDV P8-, Rab27a-, CD63-, or Rab5-specific IgG served as the primary antibody and goat anti-rabbit IgG-peroxidase served as the secondary antibody. The band intensities of western blot assay were quantified with ImageJ software.
To examine the release of exosomes from salivary glands, more than 80 viruliferous or nonviruliferous leafhoppers prepared as described above were fed on two healthy rice seedlings (approximately 10 cm in height) for 2 days. The tested plants were then collected separately and subjected to protein extraction in equal quantities. Equal amounts of proteins from each group were loaded for western blot assay.

Total RNA was extracted from roots, culms, leaves, inflorescences with different lengths, developing seeds, and all floral organs of the wild-type and m34-z mutant using the RNeasy Plant Mini Kit (Axygen). cDNA was obtained by reverse transcription using the SuperScript III Reverse Transcriptase Kit (Invitrogen) with genomic DNA digestion (Takara) using 2 μg total RNA in a 25 μL reaction volume. qPCR was carried out with the StepOne-Plus System (Applied Biosystems) using the SYBR Green PCR Master Mix kit (Promega). At least three biological replicates were performed for each tissue.