Reverse Transcription:Article Title: N(pro) of classical swine fever virus is an antagonist of double-stranded RNA-mediated apoptosis and IFN-alpha/beta induction.
Article Snippet: .. The 20 Al RT mixes contained RNA from approximately 10,000 cells in 1 TaqMan Buffer A, 5.5 mM MgCl2, 500 AM of each dNTP, 2.5 AM random hexamers, 0.8 U/Al RNasin (Promega) and 1.25 U/Al MultiScribe reverse transcriptase and were kept for 10 min at room temperature followed by incubation at 48 -C for 30 min and enzyme inactivation at 95 -C for 10 min. Then, 4 Al of the RT reaction or of the no RT control reaction was added to the PCR mix consisting of 1 TaqMan Buffer A, 4.5 mM MgCl2, 200 AM of each dNTP, 400 nM of the respective sense and antisense primer, 100 nM of the corresponding dual-labeled TaqMan probe and 0.02 U/Al AmpliTaq Gold DNA polymerase in a final volume of 25 Al. .. The real-time PCR was performed in a ABI PRISM 7700 Sequence Detection System (Applied Biosystems) with the following cycling profile: 10 min at 95 -C (activation of the AmpliTaq Gold DNA polymerase) followed by 50 cycles of 15 s at 95 -C and 60 s at 60 -C. The IFN-h mRNA induction was calculated by subtracting the mean CT value obtained for the RT-PCR from the CT value of the no RT control PCR (residual DNA).
Incubation:Article Title: N(pro) of classical swine fever virus is an antagonist of double-stranded RNA-mediated apoptosis and IFN-alpha/beta induction.
Article Snippet: .. The 20 Al RT mixes contained RNA from approximately 10,000 cells in 1 TaqMan Buffer A, 5.5 mM MgCl2, 500 AM of each dNTP, 2.5 AM random hexamers, 0.8 U/Al RNasin (Promega) and 1.25 U/Al MultiScribe reverse transcriptase and were kept for 10 min at room temperature followed by incubation at 48 -C for 30 min and enzyme inactivation at 95 -C for 10 min. Then, 4 Al of the RT reaction or of the no RT control reaction was added to the PCR mix consisting of 1 TaqMan Buffer A, 4.5 mM MgCl2, 200 AM of each dNTP, 400 nM of the respective sense and antisense primer, 100 nM of the corresponding dual-labeled TaqMan probe and 0.02 U/Al AmpliTaq Gold DNA polymerase in a final volume of 25 Al. .. The real-time PCR was performed in a ABI PRISM 7700 Sequence Detection System (Applied Biosystems) with the following cycling profile: 10 min at 95 -C (activation of the AmpliTaq Gold DNA polymerase) followed by 50 cycles of 15 s at 95 -C and 60 s at 60 -C. The IFN-h mRNA induction was calculated by subtracting the mean CT value obtained for the RT-PCR from the CT value of the no RT control PCR (residual DNA).
Control:Article Title: N(pro) of classical swine fever virus is an antagonist of double-stranded RNA-mediated apoptosis and IFN-alpha/beta induction.
Article Snippet: .. The 20 Al RT mixes contained RNA from approximately 10,000 cells in 1 TaqMan Buffer A, 5.5 mM MgCl2, 500 AM of each dNTP, 2.5 AM random hexamers, 0.8 U/Al RNasin (Promega) and 1.25 U/Al MultiScribe reverse transcriptase and were kept for 10 min at room temperature followed by incubation at 48 -C for 30 min and enzyme inactivation at 95 -C for 10 min. Then, 4 Al of the RT reaction or of the no RT control reaction was added to the PCR mix consisting of 1 TaqMan Buffer A, 4.5 mM MgCl2, 200 AM of each dNTP, 400 nM of the respective sense and antisense primer, 100 nM of the corresponding dual-labeled TaqMan probe and 0.02 U/Al AmpliTaq Gold DNA polymerase in a final volume of 25 Al. .. The real-time PCR was performed in a ABI PRISM 7700 Sequence Detection System (Applied Biosystems) with the following cycling profile: 10 min at 95 -C (activation of the AmpliTaq Gold DNA polymerase) followed by 50 cycles of 15 s at 95 -C and 60 s at 60 -C. The IFN-h mRNA induction was calculated by subtracting the mean CT value obtained for the RT-PCR from the CT value of the no RT control PCR (residual DNA).
Polymerase Chain Reaction:Article Title: N(pro) of classical swine fever virus is an antagonist of double-stranded RNA-mediated apoptosis and IFN-alpha/beta induction.
Article Snippet: .. The 20 Al RT mixes contained RNA from approximately 10,000 cells in 1 TaqMan Buffer A, 5.5 mM MgCl2, 500 AM of each dNTP, 2.5 AM random hexamers, 0.8 U/Al RNasin (Promega) and 1.25 U/Al MultiScribe reverse transcriptase and were kept for 10 min at room temperature followed by incubation at 48 -C for 30 min and enzyme inactivation at 95 -C for 10 min. Then, 4 Al of the RT reaction or of the no RT control reaction was added to the PCR mix consisting of 1 TaqMan Buffer A, 4.5 mM MgCl2, 200 AM of each dNTP, 400 nM of the respective sense and antisense primer, 100 nM of the corresponding dual-labeled TaqMan probe and 0.02 U/Al AmpliTaq Gold DNA polymerase in a final volume of 25 Al. .. The real-time PCR was performed in a ABI PRISM 7700 Sequence Detection System (Applied Biosystems) with the following cycling profile: 10 min at 95 -C (activation of the AmpliTaq Gold DNA polymerase) followed by 50 cycles of 15 s at 95 -C and 60 s at 60 -C. The IFN-h mRNA induction was calculated by subtracting the mean CT value obtained for the RT-PCR from the CT value of the no RT control PCR (residual DNA).
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