Total RNAs were extracted from the retinal tissues using RNApure Total RNA Extraction kit (BioTeke, Beijing, China) and reverse-transcribed to cDNAs using M-MLV Reverse Transcriptase (BioTeke). The volume of obtained cDNAs was 20 µl. qPCR analysis was carried out following the reaction conditions on Real-Time PCR system (BIONEER, Daejeon, Korea): 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec. The primers used were as follows: NgR forward, 5′-GTCCCTTCCAGACCAATCAGC-3′ and reverse, 5′-GCCATTGCCTGGTGGAGTGT-3′; RhoA forward, 5′-TCGGAATGATGAGCACACAA-3′ and reverse, 5′-GCTTCACAAGATGAGGCAC-3′; Rock1 forward, 5′-GTGATGGCTATTATGGACG-3′ and reverse, 5′-AGGAAGGCACAAATGAGAT-3′; F-actin forward, 5′-GAAGAGAAAGCAGCAGTGTTA-3′ and reverse, 5′-GGAGCCAGAGGGTGGTTAT-3′; GAP-43 forward, 5′-AGGGAGATGGCTCTGCTAC-3′ and reverse, 5′-CACATCGGCTTGTTTAGGC-3′; GAPDH forward, 5′-CGGCAAGTTCAACGGCACAG-3′ and reverse, 5′-CGCCAGTAGACTCCACGACAT-3′. The primers were synthesized by Sangon Biotech. Gene expression was normalized to GAPDH and calculated using the 2−ΔΔCq formula.
Real time pcr system
Manufactured by Bioneer
Sourced in China, Japan
The Real-time PCR system is a laboratory instrument used for the amplification and detection of specific nucleic acid sequences in real-time. It allows for the quantification of DNA or RNA targets by monitoring the fluorescence signal generated during the PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by BioTeke (1 mentions)
Rnapure total rna extraction kit, by BioTeke (1 mentions)
Taq pcr mastermix, by Solarbio (1 mentions)
Sybr green, by Solarbio (1 mentions)
Beyort 2 m mlv reverse transcriptase, by Beyotime (1 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
4 protocols using real time pcr system
1
Retinal RNA Extraction and qPCR Analysis
2
Reverse Transcription qPCR for Rat Lung and THP-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT-qPCR protocols follow previous reference [30 (link)] [PMID: 30,391,287]. TRIpure (BioTeke Bio., Beijing, China) method was adopted to extract total RNA from rat lung tissues or THP-1 cells. The extracted-RNA was reversely transcribed into cDNA by BeyoRT II M-MLV reverse transcriptase (Beyotime Biotech Co., Ltd., Shanghai, China). RT-qPCR was conducted with 2× Taq PCR MasterMix (Solarbio, Beijing, China) and SYBR green (Solarbio, Beijing, China) on a real-time PCR system (Bioneer Corporation, Daejeon, Korea). The data were analyzed with the 2−ΔΔCt method. GAPDH was used as internal control. The gene primers were presented as follows: COX-2: F: 5’-GAA CAC GGA CTT GCT CAC TT-3’, R: 5’-ACG ATG TGT AAG GTT TCA GG-3’; iNOS: F: 5’-TTG GAG CGA GTT GTG GAT TG, R: 5’-GTG AGG GCT TGC CTG AGT GA-3’.
3
Total RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific). cDNAs were synthesized from total RNA (2 μg) using 2 × cDNA Synthesis Master Mix (LeGene, San Diego, CA, USA), and PCR was performed with 2 × Direct MasterMix (Koma Biotech, Seoul, Korea). To quantitatively estimate gene expression, quantitative PCR was performed with 2 × SYBRGreen Mix (Koma Biotech), and the results were analyzed using a real-time PCR system (Bioneer, Daejeon, Korea). The PCR primers are listed in Supplementary Table 1 .
4
Profiling miR-21 and miR-214 in Prostate Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression level of miR-21 and miR-214 were measured for 70 prostate cancer patients (32 metastatic and 38 non-metastatic) and 30 healthy participants using Sybr Green Master mix (Takara; Japan) by using Real-time PCR system (Bioneer; South Korea). Urine samples were normalized to internal standard control U6snRNA. This method was chosen for its precision, high sensitivity and low cost. The forward and reverse primer sequences for miR-21, miR-214 and internal reference (U6SnRNA) were designed (Table1).
The reaction was carried out in a volume of 14 μL which included 7 μL of Sybr Green Master mix, 1 μL primer (10 pmol), 3μL cDNA diluted 1 to 4 and 3 μL of distilled water.
The Real-time PCR conditions was as follows: Polymerase activation at 95°C for 12 min, denaturation at 95°C for 15 sec, annealing 60°C for 30 sec and extension at 72°C for 15 sec for 42 cycles. The experiment was repeated three times. For each microRNA and U6snRNA, no template control (NTC) was provided which lacked a template (cDNA) for reproduction. PCR performance was calculated using the standard curve and serial dilutions of 107%. Finally, 2% agarose gel electrophoresis was used to ensure the reproduction and speci city of the components.
Table 1. Primer Sequences of miR-21, miR-214 and U6SnRNA.
The reaction was carried out in a volume of 14 μL which included 7 μL of Sybr Green Master mix, 1 μL primer (10 pmol), 3μL cDNA diluted 1 to 4 and 3 μL of distilled water.
The Real-time PCR conditions was as follows: Polymerase activation at 95°C for 12 min, denaturation at 95°C for 15 sec, annealing 60°C for 30 sec and extension at 72°C for 15 sec for 42 cycles. The experiment was repeated three times. For each microRNA and U6snRNA, no template control (NTC) was provided which lacked a template (cDNA) for reproduction. PCR performance was calculated using the standard curve and serial dilutions of 107%. Finally, 2% agarose gel electrophoresis was used to ensure the reproduction and speci city of the components.
Table 1. Primer Sequences of miR-21, miR-214 and U6SnRNA.
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!