RNA and proteins were extracted using Nucleospin® RNA/Protein (Macherey-Nagel, BMS, Korea) according to the manufacturer’s instructions. cDNA was synthesized using AccuPower® RT/PCR PreMix (Bioneer, Korea). qPCR was performed with SYBR qPCR Mix (CellSafe, Yongin, Korea) on a CFX96 Real-Time System (Bio-Rad, BMS, Korea). Data were analyzed with the Pfaffl method (Pfaffl, 2001 (link)) using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as reference.
Nucleospin rna protein
Manufactured by Macherey-Nagel
Sourced in Germany, France, United States
NucleoSpin RNA/protein is a lab equipment product designed for the simultaneous purification of RNA and protein from a single sample. It employs a silica-membrane technology to efficiently isolate both biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Mastercycler realplex4, by Eppendorf (3 mentions)
Real mastermix sybr green detection system, by Eppendorf (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (2 mentions)
Fast sybr green, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr mix, by Thermo Fisher Scientific (2 mentions)
Steponeplus cycler, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Accupower rt pcr premix, by Bioneer (1 mentions)
Icycler iq optical system software, by Bio-Rad (1 mentions)
Icycler thermal cycler, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Direct zol rna miniprep, by Zymo Research (1 mentions)
Rt2 qpcr primer assay, by Qiagen (1 mentions)
Stepone system, by Thermo Fisher Scientific (1 mentions)
27 protocols using nucleospin rna protein
1
Quantitative Gene Expression Analysis
2
Quantifying mRNA Expression in Human and Mouse Kidneys
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from HK-2 cells, using Direct-zol™ RNA MiniPrep supplied by Zymo Research (Irvine, CA), or mouse kidneys, using NucleoSpin RNA/Protein (Macherey-Nagel, Duren, Germany) followed by RNeasy® Mini Kit (QIAGEN, Germantown, MD). The OD ratio (optical density at 260 nm/280 nm) of RNA was always higher than 1.9. Reverse transcription was performed according to the manufacturer on 1.0 μg total RNA using iScript™ cDNA Synthesis Kit (Bio-Rad). RT2 qPCR Primer Assay [human (HK-2 cells) and mouse (kidneys) primers from QIAGEN] were used to quantify the mRNA expression of heme oxygenase 1 (HO-1) and heat shock protein 70 (Hsp70). Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (human and mouse kidney, respectively, RT2 qPCR Primer Assay from QIAGEN). Data are presented as columns, displaying mean ± SEM, for in vitro data and box plots, displaying medians and 25th and 75th percentiles, for in vivo data. The fold change values were calculated by normalizing against control samples from untreated cells or animals (controls). Expression was analyzed using iTaq™ Universal SYBR® Green Supermix (Bio-Rad). Amplification was performed as described by the manufacturer (Bio-Rad) for 40 cycles in an iCycler Thermal Cycler (Bio-Rad) and data analyzed using iCycler iQ Optical System Software (Bio-Rad).
3
Colon 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 isolated from the colon using a commercial kit (Nucleospin RNA/Protein; Macherey-Nagel, France). Proteins and mRNA are obtained from the same colon sample and not from two portions of the same sample using this kit. RNA quantification was performed by spectrophotometry (Nanodrop; Nyxor Biotech, France). Reverse transcription of mRNA was carried out in a final volume of 20 µL from 1 µg total RNA (high capacity cDNA RT kit; Applied Biosystems, Villebon Sur Yvette, France). cDNA was amplified by PCR using Fast SYBR green (Applied Biosystems) in the one-step system (Applied Biosystems). SYBR green dye intensity was analyzed using one-step software. All results were normalized to the reference gene, POLR2A [50 (link),51 (link)].
4
RNA and Protein Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA and proteins were extracted by the NucleoSpin® RNA/Protein (MACHEREY-NAGEL, Düren, Germany). The quantity and purity of RNA were determined by measuring the optical density at 260 nm with a spectrophotometer (NanoDrop Technologies, Wilmington, DE). Protein extraction Samples were maintained in PBS + 1% SDS solution at −20 °C.
5
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 using Nucleospin RNA/protein (Macherey Nagel, France) according to the manufacturer’s instructions. Reverse transcription and quantitative PCR were performed as described previously [29 (link)]. Primers used are listed in S1 Table . PCR efficiencies ranged between 93% and 100%, depending on primer pairs. For relative quantification, the -2ΔΔCt method was used. The reference gene was Gapdh and each gene was analyzed from two independent experiments performed in triplicate.
6
Quantitative Analysis of Fibroblast Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted with NucleoSpin RNA/protein (Macherey-Nagel, Duren, Germany) and quantified by NanoDrop (Thermo Scientific, Wilmington, USA), which also evaluates RNA integrity.
The qRT-PCR was performed on an Eppendorf Realplex 4 Mastercycler using the Real MasterMix SYBR Green detection system (Eppendorf, Milan, Italy) in a total volume of 10 μl loaded in triplicate. Primers for fibroblast and myofibroblast markers, fibroblast-specific protein-1 (S100A4, NM_002961) and αSMA (NM_001613), as well as COL1 (NM_000088), FN (NM_002026), CXCR4 (NM_00100854), and β-actin (NM_001101, housekeeping gene) were supplied by Primerdesign (Primerdesign, UK).
The melting curve was performed to confirm the specificity of the SYBR green assay. The qRT-PCR was performed on each independent in vitro experiment on cultures of fibrocytes isolated from all the enrolled SSc patients and HSs. Gene expression values were calculated using the comparative ΔΔCT method [18 (link)].
The qRT-PCR was performed on an Eppendorf Realplex 4 Mastercycler using the Real MasterMix SYBR Green detection system (Eppendorf, Milan, Italy) in a total volume of 10 μl loaded in triplicate. Primers for fibroblast and myofibroblast markers, fibroblast-specific protein-1 (S100A4, NM_002961) and αSMA (NM_001613), as well as COL1 (NM_000088), FN (NM_002026), CXCR4 (NM_00100854), and β-actin (NM_001101, housekeeping gene) were supplied by Primerdesign (Primerdesign, UK).
The melting curve was performed to confirm the specificity of the SYBR green assay. The qRT-PCR was performed on each independent in vitro experiment on cultures of fibrocytes isolated from all the enrolled SSc patients and HSs. Gene expression values were calculated using the comparative ΔΔCT method [18 (link)].
7
Brain Tissue Isolation and Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For brain tissue preparation, mice were deeply anesthetized with Zoletil (12.5 mg/kg) and Rompun mix (17.5 mg/kg) administered intraperitoneally. Mice were perfused transcardially with heparin dissolved in PBS (pH 7.2). The dissected brain tissues were frozen at -80 °C for Western blotting. Tissues were homogenized with total RNA and protein isolation kit (NucleoSpin® RNA/Protein #740933.50, Macherey-Nagel, Dűren, Germany). The protein samples were quantified with Pierce™ BCA Protein Assay Kit and 50 μg protein sample were used for each Western blot. The primary antibodies were applied in the following concentrations: anti-GFAP (rabbit, 1: 1,000; Dako #Z0334), anti-Iba1 (rabbit, 1:300; Wako #NB100-1028), anti-EAAT1 (rabbit, 1:1,000; Santacruz # sc-15316), anti-EAAT2 (rabbit, 1:1,000; Cellsignaling #3838), anti-Cx43 (mouse, 1:1,000; Santacruz #sc-59949), anti-PSD95 (mouse, 1:2,000; Thermo #MA1-046), anti-NeuN (mouse, 1:1,000; Millipore #MAB377) anti-GAPDH (rabbit, 1:10,000; Abfrontier #LF-PA0018). Secondary antibodies were conjugated with horse-radish peroxidase (HRP) (1: 10,000, Invitrogen). The HRP signals were visualized using an enhanced chemiluminescent (ECL, Abfrontier #LF-QC0101, Gyeonggi-do, Korea) substrate.
8
HIF-1α Protein Quantification in Gingival Tissue
9
Quantitative Analysis of TRPM2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from kidneys and cells according to manufacturer´s instruction (NucleoSpin RNA/protein, Machery & Nagel). 2 ng RNA were reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Takara, RR037A). cDNA was added to Taqman Fast Advanced master mix (Applied Biosystem, 4444556), Euk 18SrRNA (20×) (Applied Biosystem 4319413E) and TRPM2-FAM (mouse: Mm00663098 _m1, human: Hs01066086_m1), and subjected to qPCR.
10
Quantifying AMPK mRNA Levels in Skeletal Muscle
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!