Total RNA was isolated from the retina of rd10 using the RNAiso Plus kit (TAKARA Bio Inc., Japan), and the Reverse Transcriptase Superscript II Kit (TAKARA Bio Inc., Japan) was then used to prepare cDNA following the instructions. Rea-time PCR was performed in a 20-μL reaction system, containing 10 μL of 2 × SYBR Premix Ex Taq, 2 μL of cDNA, and 10 μmol/L primer pairs. The thermocycler settings were 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 34 s.
Superscript 2 reverse transcriptase kit
Manufactured by Takara Bio
Sourced in Japan, China, United States
SuperScript II reverse transcriptase kit is a laboratory product designed for the conversion of RNA to complementary DNA (cDNA). It is a key tool for various molecular biology applications that require the generation of cDNA from RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso plus kit, by Takara Bio (5 mentions)
Ab28696, by Abcam (3 mentions)
Sybr green er quantitative pcr supermix universal kit, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Tween, by Merck Group (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Aa 861, by Merck Group (1 mentions)
Rna trizol, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Abi steponeplus system, by Thermo Fisher Scientific (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Pmd19 t vector, by Takara Bio (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Poly a polymerase, by Takara Bio (1 mentions)
21 protocols using superscript 2 reverse transcriptase kit
1
Quantifying Retinal Gene Expression in rd10 Mice
2
Retinal Transcriptome and Protein Analysis
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Total RNA from rd10 retina were isolated using the RNAiso Plus kit (TAKARA Bio inc, Japan), and the Reverse Transcriptase Superscript II Kit (TAKARA Bio inc, Japan) was then used to prepare cDNA following the instructions. Real time PCR was performed in 20μL reaction system, containing10μL of 2×SYBR Premix Ex Taq, 2μL of cDNA, and 10μmol/L of the primer pairs. Thermocycler settings were: 95°C for 30s; 40 cycles of 95°C for 5s, 60°C for 34s.
Western Blot RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scienti c, MA, USA) was used to lyse homogenized retinal tissue. Samples were then spun for 10 minutes at 10,000 rpm, after which a BCA assay was used for protein quanti cation.
Equivalent protein amounts were utilized for western blotting. The primary antibodies were incubated overnight including anti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8, β-actin (ab28696, ab28696, Abcam, Cambridge, MA). After washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
Western Blot RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scienti c, MA, USA) was used to lyse homogenized retinal tissue. Samples were then spun for 10 minutes at 10,000 rpm, after which a BCA assay was used for protein quanti cation.
Equivalent protein amounts were utilized for western blotting. The primary antibodies were incubated overnight including anti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8, β-actin (ab28696, ab28696, Abcam, Cambridge, MA). After washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
3
Total RNA Isolation and qPCR Analysis
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Total RNA fromrd10 retina were isolated using the RNAiso Plus kit (TAKARA Bio inc, Japan), and the Reverse Transcriptase Superscript II Kit (TAKARA Bio inc, Japan) was then used to prepare cDNA following the instructions. Real time PCR was performed in 20μL reaction system, containing10μL of 2×SYBR Premix Ex Taq, 2μL of cDNA, and 10μmol/L of the primer pairs. Thermocycler settings were: 95°C for 30s; 40 cycles of 95°C for 5s, 60°C for 34s.
4
RNA Isolation and qPCR Analysis in Rd10 Retina
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Total RNA was isolated from the retina of rd10 using the RNAiso Plus kit (TAKARA Bio Inc, Japan), and the Reverse Transcriptase Superscript II Kit (TAKARA Bio Inc, Japan) was then used to prepare cDNA following the instructions. Rea-time PCR was performed in a 20-μL reaction system, containing 10 μL of 2×SYBR Premix Ex Taq, 2 μL of cDNA, and 10 μmol/L primer pairs. The thermocycler settings were 95°C for 30 s and 40 cycles of 95°C for 5 s and 60°C for 34 s.
Western blot analysis RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scienti c, MA, USA) was used to lyse homogenized retinal tissue. The samples were then centrifuged for 10 minutes at 10,000 rpm, after which a BCA assay was used for protein quanti cation. Equivalent amounts of protein were utilized for western blotting. The blots were incubated overnight in primary antibodies, including anti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8 and βactin (ab28696, ab28696, Abcam, Cambridge, MA). After being washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
Western blot analysis RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scienti c, MA, USA) was used to lyse homogenized retinal tissue. The samples were then centrifuged for 10 minutes at 10,000 rpm, after which a BCA assay was used for protein quanti cation. Equivalent amounts of protein were utilized for western blotting. The blots were incubated overnight in primary antibodies, including anti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8 and βactin (ab28696, ab28696, Abcam, Cambridge, MA). After being washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
5
Retinal Gene Expression Analysis
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Total RNA was isolated from the retina of rd10 using the RNAiso Plus kit (TAKARA Bio Inc, Japan), and the Reverse Transcriptase Superscript II Kit (TAKARA Bio Inc, Japan) was then used to prepare cDNA following the instructions. Rea-time PCR was performed in a 20-μL reaction system, containing 10 μL of 2×SYBR Premix Ex Taq, 2 μL of cDNA, and 10 μmol/L primer pairs. The thermocycler settings were 95°C for 30 s and 40 cycles of 95°C for 5 s and 60°C for 34 s.
Western blot analysis RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scienti c, MA, USA) was used to lyse homogenized retinal tissue. The samples were then centrifuged for 10 minutes at 10,000 rpm, after which a BCA assay was used for protein quanti cation. Equivalent amounts of protein were utilized for western blotting. The blots were incubated overnight in primary antibodies, including anti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8(sc-365042, SANTA) and β-actin (ab28696, Abcam, Cambridge, MA). After being washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
Western blot analysis RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scienti c, MA, USA) was used to lyse homogenized retinal tissue. The samples were then centrifuged for 10 minutes at 10,000 rpm, after which a BCA assay was used for protein quanti cation. Equivalent amounts of protein were utilized for western blotting. The blots were incubated overnight in primary antibodies, including anti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8(sc-365042, SANTA) and β-actin (ab28696, Abcam, Cambridge, MA). After being washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
6
Inflammatory Liver Injury Pathway
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D-Galactosamine (D-GalN), lipopolysaccharide (LPS), AA-861, dimethyl sulfoxide (DMSO), Tween, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO, USA). Dulbecco-Vogt phosphate-buffered saline (DPBS), LTB4 Elisa kit, and Sep-Pak C18 cartridges were obtained from Cayman Chemical Co. (Ann, Arbor, MI, USA). β-Mercaptoethanol, tetramethylethylenediamine (TEMED), and diethypyrocarbonate (DEPC) were purchased from Amresco Biochemical Co. ( Solon, OH, USA). RIPA and TRIzol RNA were purchased from Invitrogen (Carlsbad, CA, USA). The microbicinchoninic acid (BCA) protein assay reagent kit was from Pierce (Rockford, IL, USA). Immobilon-P transfer membranes were from Millipore (Bedford, MA, USA). Mouse anti-rat monoclonal ED1 antibody and rabbit anti-rat polyclonal 5-LO antibody were provided by Abcam (Cambridge, MA, USA). Rabbit anti-rat monoclonal β-actin antibody was purchased from Epitomics (Burlingame, CA, USA). TNF-α Elisa kit was provided by IBL (Minneapolis, MN, USA). The SuperScript II Reverse Transcriptase Kit, TaqMan Gene Expression Assay, and universal TaqMan 2x polymerase chain reaction (PCR) master mix were from Takara Bio (Dalian, China).
7
Quantifying mRNA Expression in Mouse Kidneys
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Total RNA was extracted from the kidneys of mice using TRIzol® reagent (Takara Bio, Inc.) and cDNA was synthesized in 20-µl reactions using the Superscript II reverse transcriptase kit (Takara Bio, Inc.). The expression levels of mRNAs were evaluated by qPCR with SYBR-Green reaction kit (cat. no. a25742; Applied Biosystems; Thermo Fisher Scientific, Inc.) and the ABI Step One Plus system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and normalized against the expression of the GAPDH gene The amplification conditions were as follows: 50 °C for 2 min and 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. After that, a melting curve analysis was performed. The primers used for the RT-PCR were designed and synthesized by Sangon Biotechnology Co., Ltd., and they are listed in Table 1 . The data obtained were analyzed using the 2-ΔΔCq method [20 (link)].
8
Verifying RNA-Seq Results via qRT-PCR
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Six functional genes were selected at random using qRT-PCR to verify the results of RNA-Seq (Table S3 ). The experimental materials and RNA preparation methods were the same as those used for the RNA-Seq described earlier. A SuperScript II reverse transcriptase kit (TaKaRa, Dalian, China) was used to synthesize cDNA. qRT-PCR was performed with a StepOnePlus Real-Time PCR system (2012 Life Technologies Corporation, Carlsbad, CA, USA, version 2.3). The RT-PCR program was as follows: 30 s at 95 °C; 40 cycles of 95 °C for 10 s, 60 °C for 30 s; 95 °C for 15 s–60 °C for 60 s–95 °C for 15 s was used to generate a melting curve. The reaction mixture in a 20-μL volume contained the following: 10 μL of 2 × ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 1 μL of template cDNA, 0.4 μL of each primer, and 8.2 μL ddH2O. Three replicate analyses were performed for each reaction, and then the data were analyzed using the 2−ΔΔCT method [29 (link)]. The transcript level of ThAPRT served as the internal standard (Table S3 ).
9
Quantitative Real-Time PCR Protocol
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Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. For reverse transcription, the total RNA was used with a SuperScript II reverse transcriptase kit (Takara Bio, Japan), and real-time PCR was performed using SYBR Premix EX Taq (Takara Bio) on a 7300 real-time PCR system (Applied Biosystems, USA). β-actin was used as an endogenous control and mRNA expression, relative to that in untreated cells (control), was quantified by the 2−ΔΔCT method. Primer sequences used are presented in Supplementary Table S2 .
10
Molecular Cloning and Mutagenesis of Bermudagrass Genes
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Total RNA was extracted from the stolons of the bermudagrass plants using the RNAprep pure Plant kit (TIANGEN, Beijing, China). cDNA was synthesized using the SuperScript™ II reverse transcriptase kit (Takara, Dalian, China). The coding sequence (CDS) of catalase (Cd8B1G029850.1) and malate dehydrogenase (Cd2A1G010870.1) was PCR amplified from the cDNA and cloned into the pMD™19-T vector (Takara). Site-specific mutations of the two genes were generated using overlap PCR method with sequencing-confirmed clones as templates [68 (link)]. The used primers are listed in Table S11 .
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