Polymerase Chain Reaction:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Aerosol:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Reverse Transcription:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms
Article Snippet: .. Briefly, a total volume of 10 μl containing 200 ng of total RNA, 2 μl 5X ProtoScript II reaction buffer (NEB), 25 μM ATP, 25 μM dNTPs, 50 μM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42 °C for 1 h, followed by enzyme inactivation at 95 °C for 5 min. MiRNA-specific primers were designed as previously described . cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 250 nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. ..
Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms.
Article Snippet: Real-time qRT-PCR analysis of mRNAs Total RNA was extracted using TRIZol according to the manufacturer’s instructions and dissolved in 20 – 50 μl of ultrapure water (Invitrogen), depending on RNA pellet size. .. 1 μl was reversetranscribed in a volume of 20 μl containing 4 μl of 5X ProtoScript II reaction buffer (NEB), 25 μM dNTPs, 50 μM oligo dT, 50 μM random hexamers, 2 μl DTT, 20 units of ProtoScript II reverse transcriptase (NEB), and 0.25 μl of murine RNase inhibitor (NEB). .. The reaction was incubated at 42°C for 1 h followed by enzyme inactivation at 65 °C for 15 min. cDNA was diluted 1:20 and real-time quantitative reverse transcriptase PCR (RT qPCR) was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 500 nM of each primer at the cycling conditions of 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second AR TI CL E IN P RE SS derivative maximum of the amplification curve for three technical triplicates for each sample.
Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms.
Article Snippet: .. Briefly, a total volume of 10 μl containing 200 ng of total RNA, 2 μl 5X ProtoScript II reaction buffer (NEB), 25 μM ATP, 25 μM dNTPs, 50 μM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42 °C for 1 h, followed by enzyme inactivation at 95°C for 5 min. MiRNA-specific primers were designed as previously described79. cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 250 nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95°C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60°C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. ..
SYBR Green Assay:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Real-time Polymerase Chain Reaction:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Virus:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Control:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Plasmid Preparation:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Amplification:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms
Article Snippet: .. Briefly, a total volume of 10 μl containing 200 ng of total RNA, 2 μl 5X ProtoScript II reaction buffer (NEB), 25 μM ATP, 25 μM dNTPs, 50 μM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42 °C for 1 h, followed by enzyme inactivation at 95 °C for 5 min. MiRNA-specific primers were designed as previously described . cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 250 nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. ..
Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms.
Article Snippet: .. Briefly, a total volume of 10 μl containing 200 ng of total RNA, 2 μl 5X ProtoScript II reaction buffer (NEB), 25 μM ATP, 25 μM dNTPs, 50 μM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42 °C for 1 h, followed by enzyme inactivation at 95°C for 5 min. MiRNA-specific primers were designed as previously described79. cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 250 nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95°C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60°C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. ..
Reverse Transcription Polymerase Chain Reaction:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Hood:Article Title: The Influenza A Virus M2 Protein trans -Complementation System Offers a Set of Tools for the Undergraduate Virology Laboratory
Article Snippet: Alternatively, virus samples can be extracted using Trizol and the Direct-zol kit from Zymo Research. .. RNAse-free 1.5 ml microfuge tubes RNAse-free 0.2 ml thin-walled PCR tubes Aerosol-resistant micropipette tips (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) Micropettors (0.5-10, 2-20, 20-200, and 100-1000 μl sizes) RNAse-free or DEPC-treated water 1Protoscript II reverse transcriptase and associated 5X Protoscript II reaction buffer (New England Biolabs, M0368S) dNTPs (10 mM each; New England Biolabs) PowerUp SYBR Green Master mix (ThermoFisher Scientific) Real-time PCR reaction plate or tubes Sealing film or caps INFA-F primer; 2 μM INFA-F and INFA-R primers; 40 μM Virus samples in liquid medium Control plasmid, pAD118 (Fragment of segment 7 from A/Puerto Rico/8/1934/H1N1 that contains the amplicon targeted by this RT-PCR reaction) Equipment: Level 2 biosafety cabinet 0.5-10 μl single-channel micropipette 2-20 μl single-channel micropipette 20-200 μl single-channel micropipette 100-1000 μl single-channel micropipette Centrifuge capable of centrifuging real-time PCR plates Vortex mixer Thermocycler 2Real-time PCR system capable of detecting ROX and SYBR Green. ..
Incubation:Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms
Article Snippet: .. Briefly, a total volume of 10 μl containing 200 ng of total RNA, 2 μl 5X ProtoScript II reaction buffer (NEB), 25 μM ATP, 25 μM dNTPs, 50 μM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42 °C for 1 h, followed by enzyme inactivation at 95 °C for 5 min. MiRNA-specific primers were designed as previously described . cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 250 nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. ..
Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms.
Article Snippet: .. Briefly, a total volume of 10 μl containing 200 ng of total RNA, 2 μl 5X ProtoScript II reaction buffer (NEB), 25 μM ATP, 25 μM dNTPs, 50 μM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42 °C for 1 h, followed by enzyme inactivation at 95°C for 5 min. MiRNA-specific primers were designed as previously described79. cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 250 nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95°C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60°C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. ..
Quantitative RT-PCR:Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms
Article Snippet: .. Briefly, a total volume of 10 μl containing 200 ng of total RNA, 2 μl 5X ProtoScript II reaction buffer (NEB), 25 μM ATP, 25 μM dNTPs, 50 μM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42 °C for 1 h, followed by enzyme inactivation at 95 °C for 5 min. MiRNA-specific primers were designed as previously described . cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 250 nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. ..
Article Title: The microRNA miR-71 suppresses maladaptive UPR mt signaling through both cell-autonomous and cell-non-autonomous mechanisms.
Article Snippet: .. Briefly, a total volume of 10 μl containing 200 ng of total RNA, 2 μl 5X ProtoScript II reaction buffer (NEB), 25 μM ATP, 25 μM dNTPs, 50 μM RT primer (ik44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of ProtoScript II reverse transcriptase (NEB) were incubated at 42 °C for 1 h, followed by enzyme inactivation at 95°C for 5 min. MiRNA-specific primers were designed as previously described79. cDNA was diluted 1:5 and RT qPCR was run on a LightCycler 480 II (Roche) in a total volume of 10 μl containing 5 μl SensiFAST SYBER No-ROX (Bioline Meridian Biosystems), 1 μl of cDNA and 250 nM of each primer primer (miR-35-5p was amplified with ik16/ik17, miR-71-5p with ik38/39 and miR-59-3p with ik48/49) at the cycling conditions of 95°C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60°C for 10 s, and elongation at 72 °C for 20 s. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of the amplification curve for three technical triplicates for each sample. ..
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