T7 sp6 rna transcription kit

Double fluorescence in situ hybridization (dFISH) was performed in the gut of viruliferous planthopper. Prehybridization solution contained biotin-labeled miR-263a probe (1 μmol mL^-1; RiboBio) and digoxigenin (DIG)-labeled RNA1 3’-EUTR probe (50 ng μL^-1) for hybridization. Approximately 200 bp of the RNA probe for 3’-EUTR of RNA1 was synthesized by a T7/SP6 RNA transcription kit (Roche). The gut was dissected from viruliferous fourth-instar nymphs and fixed in 4% (wt vol^-1) paraformaldehyde at 4°C overnight. After washing with PBST, the gut was digested with proteinase K (20 μg mL^-1; Tiangen) at 37°C for 15 min and hybridized with the miRNA probe and viral RNA probe at 37°C overnight. The midgut was washed with 0.2× SSC at 37°C to remove nonspecific binding. An anti-DIG alkaline phosphatase-conjugated antibody (1:500) and an anti-biotin antibody (1:100) were used for signal detection. Probes for cel-miR-67-3p and rice PsbP gene (KF460579) were used in the control group. The fluorescent signal of DIG or biotin was generated by HNPP/Fast Red or fluorescein-tyramide (Perkin-Elmer, Waltham, MA, USA) and recorded using a Leica TCS SP5 confocal microscope (Leica Microsystems). Primers used for probe synthesis are listed in S1 Table.

The localization of RSV genomic RNAs in planthopper salivary glands and guts were performed using an RNA3 probe labeled with digoxigenin (DIG). The probe for RNA3 was synthetized using a T7/SP6 RNA Transcription kit (Roche, Basel, Switzerland) and was subsequently fragmented to approximately 250 bp via the alkaline lysis method. The primers used for RNA3 probe synthesis are listed in Table S3. Salivary glands and guts were dissected from viruliferous planthoppers and then fixed in 4% (w/v) paraformaldehyde at 4 °C overnight. After being digested with 20 μg/mL of proteinase K at 37 °C for 15 min, tissues were hybridized with 5 ng/μL of RNA3 probe at 37 °C overnight and then successively washed in 2×, 1×, and 0.2× SSC at 37 °C for 30 min twice. An anti-DIG alkaline phosphatase-conjugated antibody (1:500) was used for RNA3 probe detection. The DIG fluorescent signal was detected under an HNPP Fluorescent Detection Set (Roche). The samples without the treatment of RNA3 probe were used as negative controls. Images were viewed under a Leica TCS SP5 confocal microscope (Leica).

The RNA probe for Actβ was synthesized by a T7/SP6 RNA Transcription Kit (Roche) and was subsequently fragmented to approximately 250 bp by carbonate buffer. The primers used for probe synthesis of Actβ are in S1 Table. Ovarioles were separated from ovaries in locust saline and fixed in 4% (wt/vol) paraformaldehyde overnight. After digestion with proteinase K (20 μg/mL; Tiangen) at 37°C for 15 min, these ovarioles were hybridized with miR-34a probe (2 pmol/mL) and Actβ probe (5 ng/μL) at 37°C overnight. Then, the ovarioles were successively washed in 2× SSC, 1× SSC, and 0.2× SSC at 37°C. Anti-DIG alkaline phosphatase-conjugated antibody (1:100) and anti-biotin antibody (1:100) were used for probe detection. Then, the fluorescent signal of digoxigenin (DIG) or biotin was obtained by HNPP/Fast Red (HNPP Fluorescent Detection Set, Roche) or Fluorescein-Tyramide (TSA Fluorescein System, Perkin Elmer). Images were captured on an LSM 710 confocal fluorescence microscope (Carl Zeiss) at a magnification of 63×.