Left ankles were dissected under sterile conditions, frozen in liquid nitrogen, and then stored at −80 °C until extraction. Tissue lysis was performed in TRI Reagent (Sigma, Saint Louis, MO, US). RNA was separated from proteins after centrifugation in chloroform. RNA was purified with RNeasy plus (Qiagen, Venlo, Netherlands). Quality and quantity of RNA were assessed by Experion RNA analysis (BioRad, Hercules, CA, US) and QuantIT RiboGreen RNA assay (Thermo Scientific, Waltham, MA, US), respectively. Reverse transcription (RT) was performed on 2 µg of RNA. Quantitative RT polymerase chain reaction (PCR) was conducted on CFX96 RealTime System (BioRad) with LightCycler FastStart DNA Master plus SYBRgreen I (Roche Diagnostics, Basel, Switzerland). Primer sequences of candidate genes are detailed in Supplementary Table S1 . The results were normalized to the housekeeping gene expression hypoxanthine-guanine phosphoribosyltransferase and presented as the variation in fold of gene expression compared to CTRL group at day 0.
Experion rna analysis
Manufactured by Bio-Rad
Sourced in United States
The Experion RNA analysis system is a microfluidic-based automated electrophoresis platform that provides rapid and automated analysis of RNA samples. The system automatically separates and detects RNA molecules, generating an electropherogram and data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quant it ribogreen rna assay, by Thermo Fisher Scientific (3 mentions)
Cfx96 real time system, by Bio-Rad (3 mentions)
Lightcycler faststart dna masterplus sybr green 1, by Roche (3 mentions)
Rneasy plus, by Qiagen (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Allprep rna protein kit, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Rq1 rnase free dnase product, by Promega (1 mentions)
M mlv reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Nzyol reagent, by NZYTech (1 mentions)
4 protocols using experion rna analysis
1
Quantitative Gene Expression Analysis
2
Comprehensive RNA Extraction and Quantification
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For animal tissues (spleen, gut and ankle) and synovial organoids lysis was performed in TRI Reagent (Sigma). To obtain enough RNA, 3 synovial organoids were pooled for lysis. RNA was purified with RNeasy plus (Qiagen Inc.). For cell culture, RNA was extracted using Allprep RNA/protein kit (Qiagen Inc.). Quality and quantity of RNA were assessed by Experion RNA analysis (BioRad) and QuantIT RiboGreen RNA assay (Thermo Fisher Scientifc), respectively. Complementary DNA (cDNA) was synthesized using the iscript cDNA synthesis kit (Biorad). Quantitative RT polymerase chain reaction (PCR) was conducted on CFX96 RealTime System (BioRad) with LightCycler FastStart DNA Master plus SYBRgreen I (Roche Diagnostics, Basel, Switzerland). The results were normalized to the housekeeping gene expression hypoxanthine-guanine phosphoribosyltransferase (HPRT).
3
RNA Extraction and RT-qPCR Analysis
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RNA was extracted using a Allprep RNA/protein kit (Qiagen Inc.). Quality and quantity of RNA were assessed by an Experion RNA analysis (BioRad) and QuantIT RiboGreen RNA assay (Thermo Fisher Scientifc), respectively. Complementary DNA (cDNA) was synthesized using the iscript cDNA synthesis kit (Biorad). Quantitative RT polymerase chain reaction (PCR) was conducted on CFX96 RealTime System (BioRad) with LightCycler FastStart DNA Master plus SYBRgreen I (Roche Diagnostics, Basel, Switzerland). The results were normalized to the housekeeping gene expression hypoxanthine-guanine phosphoribosyltransferase (HPRT).
4
Sperm RNA Extraction and Analysis
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Total RNA was extracted from sperm pools of: control groups AB (n = 4) and Tg(ins:nfsb-mCherry) (n = 7) and Met-treated groups AB (n = 4) and Tg(ins:nfsb-mCherry) (n = 7). Each pool contained sperm from 5 males. The sperm sample of each male was collected, diluted in 10 µL of phosphate-buffered buffer (PBS), and added to NZYol reagent (NZYTech, Lisbon, Portugal), according to the manufacturer’s specifications. DNAse treatment was performed using RQ1 RNase-Free DNase product (Promega, Madison, WI, USA) to remove genomic DNA contamination. The purity of the RNA samples was evaluated. The concentration and purity of the total RNA samples were evaluated using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA) and the 260/280 ratios were 1.8–2.0. The integrity of the obtained RNA was assessed through Experion RNA analysis (Biorad, Hercules, CA, USA). Complementary DNA (cDNA) was synthesized from 500 ng of the total RNA using the M-MLV reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA) with an oligo (dT) primer following the manufacturer’s protocol. The reverse transcription conditions were 37 °C for 1 h followed by 70 °C for 15 min, and the samples were stored at −20 °C until further analysis.
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