Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation


Reverse transcription quantitative PCR



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Reverse transcription quantitative PCR
The cDNA was synthesized using Moloney murine leu-
kemia virus (M-MLV) kit (28025013; ThermoFisher, 
Waltham, MA, USA). A Real-time system (q-PCR) was 
used to detect PPV interrelated mRNA, β-actin as an 
internal control. All primer in Table 
1
 were synthesized 
(Sangon Biotech, Shanghai, China).
Immunofluorescence assay and confocal imaging
Immunofluorescence assay was operated as previously 
described [
32
]. The cells were incubated with the pri-
mary antibodies 3C9, SYNCRIP or Cy3-NS1 mRNA Fish 
probe. After further washing, the cells were incubated 
with the secondary antibodies for 1 h at room tempera-
ture in the dark. After the final washing, the cover glasses 
were removed from the wells and fixed onto a glass slide, 
and the image was captured by a confocal microscopy.
RNA‑immunoprecipitation assay
These assays were done according to the Abcam RNA-
immunoprecipitation manufacturer. Briefly, cells were 
harvested by trypsinization and resuspended in PBS, 
2 mL freshly prepared nuclear isolation buffer was 
added (1.28 M sucrose, 40 mM Tris–HCl pH 7.5, 20 mM 
MgCl
2
, 4% Triton X-100) and the sample was kept on ice 
for 20 min. Then the cells were centrifuged at 2500 g for 
15 min to obtain a pelleted nuclei. The nuclear pellet was 
resuspended in freshly prepared RIP buffer (150 mM KCl, 
25 mM Tris pH 7.4, 5 mM EDTA, 0.5% NP40), 1 mM 
PMSF, 100 U/mL RNase inhibitor, as well as 1 × protease 
inhibitor cocktail (Sigma). Centrifugation was then used 
to obtain a pellet containing the nuclear membrane and 
debris at 13 000 g for 10 min. Then the specific antibody 
and control antibody IgG were added for 4 h at 4 ℃, then 
40 μL protein G beads were added for 2 h. After centrifu-
gation to discard the supernatant, the beads were washed 
three times with the RIP buffer. The RNA co-precipitated 
with SYNCRIP was obtained by resuspending the beads 
in Trizol RNA extraction reagent and further extraction, 
and then was analyzed by PCR; GAPDH served as a neg-
ative control.

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