A trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to isolate the total RNA from the BV-2 microglial cells and N27-A cells following the manufacturer’s guidelines. A first-strand cDNA synthesis was performed using 2.5 μg of total RNA with the ReverTra Ace-α kit (Toyobo, Osaka, Japan). The reaction was executed at 60°C for 60 min and was then heated at 95 °C for five min; 1 μL from each RT-reaction mixture was used for PCR amplification. PCR amplification was accomplished using specific primers (Bioneer, Daejeon, Korea), as reported in Table 1 . By using 1.2% agarose-gel electrophoresis, all of the PCR products were resolved and visualized with the GelRed® Nucleic Acid Gel Stain (Biotium, Inc., Landing Parkway, Fremont, CA, USA). The gels were photographed; the pixel intensity for each band was determined in Image J (NIH), and they were normalized to the band intensity of GAPDH mRNA.
Specific primers
Manufactured by Bioneer
Sourced in United Kingdom, United States
Specific primers are short DNA sequences designed to target and amplify specific regions within a larger DNA molecule. They serve as essential tools in various molecular biology techniques, enabling the selective amplification of target genetic sequences for analysis, detection, or further experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Takara Bio (3 mentions)
Revertra ace α kit, by Toyobo (2 mentions)
G box ef imaging system, by Syngene (2 mentions)
Geneamp pcr system 2700, by Thermo Fisher Scientific (2 mentions)
Reverse transcriptase premix, by Elpis Biotech (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Gelred nucleic acid gel stain, by Biotium (1 mentions)
Rna bee isolation kit, by Tel-Test (1 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Simpliamp, by Thermo Fisher Scientific (1 mentions)
Loading star, by Dyne Bio (1 mentions)
Genesnap program, by Syngene (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Lightcycler instrument, by Roche (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix, by Bio-Rad (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Oligo dt primer, by Bioneer (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
12 protocols using specific primers
1
Quantifying Gene Expression in Microglia and Neuronal Cells
2
RNA Isolation and cDNA Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RNA-Bee isolation kit (Tel-Test, Friendswood, TX, USA) was used according to the manufacturer’s recommendations for isolating total RNA from HaCaT cells. cDNA was prepared using reverse-transcriptase M-MuLV (Fermentas Life Science, Pittsburgh, PA, USA). PCR amplification was performed using specific primers (Bioneer, Daejeon, Korea).
3
Isolation and Quantification of Muscle RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from the GA and the SOL muscles were isolated using the Trizol reagent (Takara, Otsu, Japan). Purity and concentration of RNA were determined spectrophotometrically using the NanoDrop Lite spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA was reverse-transcribed to cDNA with the RT premix at 42 ℃ for 60 min and then at 95 ℃ for 5 min using the SimpliAmp thermal cycler (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed with the synthesized cDNA, the SafeDry Taq PCR premix (CellSafe, Gyeonggi, Korea), and specific primers (Bioneer, Daejeon, Korea) (Table 1 ). Amplification steps comprised 34–36 cycles as follows: Denaturation for 30 s at 95 ℃, annealing for 30 s at 58–59 ℃, and extension for 45 s at 72 ℃. The final step included extension for 5 min at 72 ℃. After that, the amplified products stained with the Loading star (DyneBio, Daejeon, Korea) were separated on 1.5% agarose gel and detected with the G:BOX EF imaging system (Syngene, Cambridge, UK) and the Gene Snap program (Syngene). The Image J software (National Institutes of Health) was used for densitometry analysis. The β-actin served as the internal control.
4
Quantifying CCL17 and CCL22 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from samples of treated HaCaTs using BioZol reagent (Bio-Solution, Seoul, Korea) according to the manufacturer’s recommendations. cDNA was established using reverse-transcriptase M-MuLV (Fermentas Life Science, Pittsburgh, PA, USA). PCR amplification was confirmed using specific primers as follows (Bioneer, Daejeon, Korea): CCL17 (forward) 5′-ACT GCT CCA GGG ATG CCA TCG TTT TT-3′, (reverse) 5′-ACA AGG GGA TGG GAT CTC CCT CACTG-3′, CCL22 (forward) 5′-AGG ACA GAG CAT GGC TCG CCT ACA GA-3′, and (reverse) 5′- TAA TGG CAG GGA GGT AGG GCT CCT GA-3′. GAPDH mRNA was used as an internal control.
5
RT-qPCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were isolated using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA). The cDNAs were amplified by PCR using the synthesized specific primers (Bioneer, Daejeon, Korea) (Table 1 ).
RT-qPCR was operated with the lightcycler TM instrument (Roche Applied Sciences, Indianapolis, IN, USA) according to the manufacturer’s protocol. PCR started at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 57 °C for 15 s, and 72 °C for 20 s. The mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the expression of genes of interest. Each sample was tested in triplicate, and relative gene expression data were analyzed by means of the 2−ΔCT method.
RT-qPCR was operated with the lightcycler TM instrument (Roche Applied Sciences, Indianapolis, IN, USA) according to the manufacturer’s protocol. PCR started at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 57 °C for 15 s, and 72 °C for 20 s. The mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the expression of genes of interest. Each sample was tested in triplicate, and relative gene expression data were analyzed by means of the 2−ΔCT method.
6
Total RNA Extraction and RT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA isolation from the cells and RT-PCR analysis were performed using TRIzol reagent (Takara, Japan) and the Gene Amp PCR System 2700 (Applied Biosystems, USA), respectively. Isolated total RNA was quantified using the Nanodrop 1000 spectrophotometer (Thermo Scientific, USA). The cDNA was synthesized with Reverse Transcriptase Premix (Elpis-Biotech) and total RNA at 42°C for 55 min and 70°C for 15 min. PCR was carried out with SafeDry Taq PCR premix (CellSafe, Korea) and specific primers (Bioneer, Korea) of which sequences are shown in Table 1 . Thermal profile for target gene amplification was as follows: 95°C for 10 min for enzyme activation and 35 cycles of denaturing at 95°C for 30 s, annealing at 58-60°C for 30 s, and elongating at 72°C for 45 s. The amplified cDNA was stained with 5× Loading STAR dye (DyneBio, Korea) and separated by electrophoresis with 1.5% agarose gel. The cDNA band was identified using the G:BOX EF imaging system (Syngene). The band densities were quantified using the Image J software (National Institutes of Health).
7
Quantitative RT-PCR Analysis of Microglial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was extracted from the BV-2 microglial cells using a Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. A first-strand cDNA synthesis was performed using 2.5 μg of total RNA and the ReverTra Ace-α kit (Toyobo, Osaka, Japan). The reaction was performed at 60 °C for 60 min and was heated at 95 °C for 5 min; 1 μL from each RT-reaction mixture was used for a PCR amplification. The PCR amplification was performed using specific primers (Bioneer, Daejeon, Korea), as reported previously [5 (link)]. All of the PCR products were resolved using 1.2% agarose-gel electrophoresis and were visualized with ethidium bromide. For quantification, the gels were photographed, the pixel intensity for each band was determined in Image J (NIH), and they were normalized to the band intensity of GAPDH mRNA.
8
RT-PCR Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were isolated from cells using TRIzol (Thermo Fisher Scientific), and cDNAs were synthesized using RT PreMix (Bioneer, Daejeon, South Korea) and the oligo dT primer (Bioneer) according to the manufacturers’ instruction. Gene fragments were amplified using 25 ng of cDNA, specific primers (Bioneer), and PCR PreMix (Bioneer) in a total reaction volume of 20 μl. All RT-PCR amplifications were verified to be in the linear range. Real-time PCR was performed using a CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR Green Supermix (Bio-Rad). The housekeeping gene GAPDH was used as an internal standard. The primer sequences are listed in Supplementary Table S2 .
9
Quantification of SEPP1 and Fetuin-A mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the cells using Trizol reagent (Invitrogen) to measure the messenger RNA (mRNA) levels of the SEPP1 and fetuin-A genes. Total RNA (2 µg) was reverse-transcribed to complementary DNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). After cDNA synthesis, quantitative real-time PCR was performed using SYBR green (Roche, Lewis, UK) and specific primers (Bioneer Co., Daejeon, Korea) according to the manufacturers' instructions. To normalize the expression of the target genes, the expression of β-actin (Actb) was used as an endogenous control in the comparative Ct method (2-delta delta Ct).
10
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using PureLink RNA Mini kit (Invitrogen) according to the manufacturer's instructions. RNA concentration was measured on Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). Reverse transcription was performed using High Capacity cDNA Reverse Transcription kit (Invitrogen). Power SYBR Green PCR Master Mix (Applied Biosystems, Forester city, CA, USA), cDNA and specific primers (Bioneer, Daejeon, Korea) were mixed. Real-time PCR was performed using StepOnePlus (Life Technologies, Carlsbad, CA, USA). Relative expression levels were determined based on the Ct values and normalized to Ct values for the 18S gene.
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!