Establishment Of RNAscope In Situ Hybridization For Detection Of Prrsv And Its Preliminary Application

RNAscope in situ hybridization(RNAscope~?)is a novel technique based on the principle of complementary base pairing.The unique probe structure,signaling cascade and background suppression make this method innovativeness.We successfully detected porcine reproductive and respiratory syndrome virus(PRRSV)in tissue sections by RNAscope~?and testified the specificity of this technique.Subsequent quantitative analysis of RNAscope~?,in situ hybridization(ISH)and immunohischemistry(IHC)detecting PRRSV in consecutive tissue sections respectively confirmed higher sensitivity of RNAscope~?.The image analysis revealed RNAscope~?and ISH showsd dense positive signals while IHC showed less signals in thymus.In addition,RNAscope~?performed more positive signals than IHC in the same position of lymph nodes.RNAscope~?showed the most significant increase of positive signals,followed by IHC and ISH.The statistical results indicated the sensibility of RNAscope~?is significant higher than ISH and IHC.Then we established RNAscope~?and IHC double-staining technique to develop these two methods.We detected slides with RNAscope~?for PRRSV and IHC for macrophages.Results showed positive signals colocalized in different tissues as well as some sporadic non-colocalized signals.These results indicate this double-staining technique is a feasible method in detection.On account of the fact that PRRSV infection can induce cell apoptosis,we performed double-staining on slides using RNAscope~?for apoptosis regulatory gene(Bid and Bcl-xl)and IHC for PRRSV,the results showed increase tendency of Bid gene and decrease tendency of Bcl-xl after PRRSV infection.These observations confirmed that this two techniques could be used simultaneously and might be a new strategy for mechanistic research studies.In conclusion,our results confirmed the highly sensitivity and specificity of RNAscope~?in detection of PRRSV.Furthermore,the positive results of the double-staining demonstrate that combining RNAscope~?with IHC is sensitive not only in indetection of virus and cells,but also in detection of endogenous gene and viru protein.This results revealed RNAscope~?-IHC double staining could be a new strategy and platform for basic research and mechanistic studies of mixed infections or other veterinary research.