Total RNA (5 µg) was reverse-transcribed using the SuperScript III First-strand synthesis kit, as has been described previously (Islam et al. 2015 (link); Islam, Ahmed et al. 2018 (link)). The synthesized cDNA was incubated with RNase H at 37°C for 1 h. PCR was performed using 2 μL of cDNA and the following primers: following primers: IL-6 Forward 5′-GGTACATCCTCGACGGCATCT-3′ and Reverse 5′-GTGCCTCTTTGCTGCTTTCAC-3′ and IL-1β Forward 5′-ACAGATGAAGTGCTCCTTCCA-3′ and Reverse 5′-GTCGGAGATTCGTAGCTGGAT-3′ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5ʹ-AGGGCTGCTTTTAACTCTGGT-3ʹ and GAPDH reverse, 5ʹ-CCCCACTTGATTTTGGAGGGA-3ʹ. PCR was performed under the following conditions: one cycle at 98°C for 3 min followed by 30–35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension step at 72°C for 5 min. The amplified PCR products were analyzed via 2% agarose gel electrophoresis and EcoDye™ Nucleic Acid Staining Solution (Biofact Co., Ltd.); the relative intensities of the detected bands were measured on a Gel Doc2000 scanner (Bio-Rad, Hercules, CA, USA).
Ecodye nucleic acid staining solution
Manufactured by Biofact
Sourced in United States
EcoDye™ Nucleic Acid Staining Solution is a dye used for the detection and quantification of nucleic acids, including DNA and RNA, in various applications such as gel electrophoresis, fluorescence microscopy, and flow cytometry. It is a sensitive and environmentally friendly alternative to traditional nucleic acid stains.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc2000 scanner, by Bio-Rad (2 mentions)
Pgem t easy vector, by Promega (1 mentions)
Proflex pcr system, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
100 bp dna ladder, by Solgent (1 mentions)
Proflex base thermal cycler, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio design analysis software v1, by Thermo Fisher Scientific (1 mentions)
Rneasy r mini kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
6 protocols using ecodye nucleic acid staining solution
1
Quantitative RT-PCR Analysis of Inflammatory Genes
2
Chromatin Relaxation Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MNase assays were performed as previously described [39 (link)]. MNase-digested chromatin DNA was electrophoresed on 1.5% agarose gels and visualized by staining with EcoDye™ Nucleic Acid Staining Solution (Biofact, ES301-1000). The genomic band intensities without (g0) and with (gc) treatment with different concentrations of MNase were quantified using Image J software (NIH, Bethesda, MD, USA). The ratio of gc/g0 was used to represent the degree of chromatin relaxation.
3
Mitochondrial COI Amplification and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The COI region was amplified using primers CO1-C02 (5′-AYTCAACAAATCATAAAG ATATTGG-3′) and CO1-C04 (5′-ACYTCRGGRTGACCAAAAAATCA-3′), developed by Che et al. [37 (link)]. Amplification was conducted in a 40 µL PCR mixture containing 0.5 μmol L—1 of each primer, Sol 2 × Taq PCR Smart Premix 1 (Solgent, Daejeon, Korea), and approximately 15 ng template DNA using the Pro Flex PCR System (Applied Biosystems, Life Technologies, Foster City, CA, USA). PCR thermal-cycling conditions were as follows: 95 °C for 5 min; 35 cycles of 1 min at 95 °C, 1 min at 45 °C, and 1 min at 72 °C; and a final extension for 5 min at 72 °C. PCR products were separated on a 1.5% agarose gel stained with Ecodye™ Nucleic Acid Staining Solution (Biofact, Daejeon, Korea) and visualized under UV light. The size of the PCR products was estimated using a 100 bp DNA ladder (Solgent).
PCR products were extracted from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and subcloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA). Subcloned DNA fragments were sequenced in both directions using T7 and SP6 primers by the Sanger method.
PCR products were extracted from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and subcloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA). Subcloned DNA fragments were sequenced in both directions using T7 and SP6 primers by the Sanger method.
4
Quantitative Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from hTERT-RPE cells treated with DMSO or BFA using RNeasyR mini kit (Qiagen). DMSO or BFA was treated 30 min before serum-restimulation at the indicated concentrations. cDNA was generated by SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Conventional RT-PCR was performed using a ProFlex™ Base Thermal Cycler (Applied Bio-systems) with the conditions of 95°C for 20 s, 62°C for 30 s, and 72°C for 45 s for a total of 25 cycles for Plk1, Dvl2, and GAPDH, followed by a 10 min final extension at 72°C. PCR products were electrophoresed in 3% agarose gel, stained with EcoDye™ Nucleic Acid Staining Solution (BIOFACT, Korea), and photographed. Real-time RT-PCR was performed in a final volume of 20 μl with 2 μl of cDNA, 10 pmol forward and 10 pmol reverse primer in 1X power SYBG green PCR master Mix (Applied Biosystems, USA) with the condition of 95°C for 15 s for denaturation, 55°C for 1 min for annealing and 72°C for 15 s extension using an QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). The expression value of each gene was normalized by that of GAPDH. Final values were calculated using the ΔΔCt method. The results were analyzed using QuantStudio™ design & Analysis software v1.4 (Applied Biosystems). All the primers used in these experiments are summarized in Supplementary Table S3 .
5
cDNA Synthesis and RT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cDNA synthesis, we reverse-transcribed the total RNA (5 µg) using the SuperScript III First-strand synthesis kit, described previously [39 (link)]. We incubated the synthesized cDNA with RNase H at 37 °C for 2 h. We performed a PCR using 2 μL of cDNA and primers that include p21 forward, 5′-GTCCGTCAGAACCCATGC-3′ and p21 reverse, 5′-GTCGAAGTTCCATCGCTCA-3′; p53 forward, 5′-CCTCACCATCATCACACTGG-3′ and p53 reverse, 5′-CCTCATTCAGCTCTCGGAAC-3′; E-cadherin forward, 5′-TTGGCTCTGC CAGGAGCCGG-3′ and E-cadherin reverse, 5′-TGTCGACCGGTGCAATCTTC-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-AGGGCTGCTTTTAACTCTGGT-3′ and GAPDH reverse, 5′-CCCCACTTGATTTTGGAGGGA-3′. We performed the PCR under the following conditions: one cycle at 98 °C for 3 min, followed by 30–35 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension step at 72 °C for 5 min. We analyzed the amplified PCR products by 1.7% agarose gel electrophoresis and EcoDye Nucleic Acid Staining Solution (Biofact). Then, we captured the images using the Wise Capture I-1000 software (Daihan Scientific, Seoul, Korea).
6
Quantification of PRPF and GAPDH mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells with TRIzol reagent (Invitrogen), and 5 μg of the total RNA was reverse transcribed using SuperScript III Reverse Transcriptase according to the manufacturer's protocol. The resulting cDNA was incubated with RNase H at 37°C for 2 h, and PCR was performed with cDNA (2 μL) and the following primers: PRPF forward, 5′-AGGGATCGAAGCTGGAAATA-3′ and PRPF reverse, 5′-TGACCTCTGAGTCATCT-GTGG-3′; GAPDH for-ward, 5′-AGGGCTGCTTTTA-ACTCTGGT-3′ and GAPDH reverse, 5′-CCCCACTTGATTTTGGAGGGA-3′. PCR was performed under the following conditions: one cycle at 98°C for 3 min, followed by 30–40 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec, with a final extension step at 72°C for 5 min. The amplified PCR products were analyzed by 2% agarose gel electrophoresis with EcoDye™ Nucleic Acid Staining Solution (Biofact Co., Ltd.), and the relative intensities of the detected bands were measured on a Gel Doc2000 scanner (Bio-Rad, Hercules, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!