Total RNA from all samples (n = 4 per condition) was isolated by use of Trizol reagent per the manufacturer’s instructions. RNA purity (260/280 ratio ≥ 1.8) and quantity were assessed by use of a BioTek Epoch Gen 5 spectrophotometer. A total of 600 μg of RNA was reverse transcribed with an Ambion RETROscript kit. Resulting cDNA was amplified by use of a Rotogene 3000 thermocycler. Reaction products were detected with validated human QuantiTect primer assays (Qiagen) (Table 1 ) in conjunction with a QuantiTect SYBRgreen (Qiagen) polymerase chain reaction kit. Relative differences in gene expression comparing between amnion MSCs and hADSCs in basal medium and differentiation medium were calculated and reported as 2^− (CtGene of interest/CtGapdh).
Epoch gen 5 spectrophotometer
Manufactured by Agilent Technologies
The Epoch Gen 5 spectrophotometer is a high-performance instrument designed for accurate and reliable UV-Vis absorbance measurements. It features a compact design, a large sample compartment, and advanced optics to deliver precise and reproducible results across a wide range of applications.
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Lab products found in correlation
Quantitect primer assay, by Qiagen (1 mentions)
Quantitect sybr green, by Qiagen (1 mentions)
Retroscript kit, by Thermo Fisher Scientific (1 mentions)
Microcentrifuge tube, by Thermo Fisher Scientific (1 mentions)
Mir 39 spike in control, by Qiagen (1 mentions)
Mirneasy serum plasma kit, by Qiagen (1 mentions)
2 protocols using epoch gen 5 spectrophotometer
1
Comparative Gene Expression Analysis of MSCs
2
Serum miRNA Extraction from Mice
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Blood was collected from mice via axillary vessels in 1.5 ml microcentrifuge tubes (Fisher), allowed to clot for 30 min at room temperature, and centrifuged at 900 × g for 10 min and 4°C. Serum layer was transferred to a new microcentrifuge tube and centrifuged for 10 min at 16,000 × g and 4°C, and the cleared supernatant was transferred to a new microcentrifuge tube without disturbing the pellet. One hundred microliter of serum sample per mouse was processed for RNA isolation using miRNeasy Serum/Plasma Kit (Qiagen) as per manufacturer's recommended protocol or stored at −80 till processing. Serum/Plasma C. elegans miR-39 Spike-In Control (Qiagen) was spiked into each sample prior to RNA purification as an internal control for miR expression profiling in serum to allow for monitoring of RNA recovery and purity, and reverse transcription efficiency. The RNA concentration and purity were determined by Qubit RNA assay broad range (Qubit RNA assay BR, Invitrogen) fluorometry. This reagent specifically binds to RNA only and does not detect DNA, protein or free nucleotides. Additionally, spectrophotometric analysis of all samples using Epoch Gen 5 spectrophotometer (Biotek) showed that all RNA samples had A260/280 ratios ≥1.8.
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