To investigate the differentiation potential of implanted rASCs in the rabbit cornea, at 28 days post-surgery, 50 mg of wounded cornea was put into 1 mL Trizol (ThermoFisher Scientific) and homogenized thoroughly in a tissue grinder (KZ-III-F, Servicebio). Then, RNA from the samples was isolated by adding chloroform and centrifugation. The upper phase was collected and isopropanol was added, followed by centrifugation. The supernatant was discarded, and the RNA pellet was eluted into 75% ethanol, and then centrifugated. After removing the supernatant, the remaining pellet was air-dried, eluted, and quantitated using a Nanodrop 2000 (ThermoFisher Scientific). Enough RNA was obtained and transcribed to cDNA by using a quantitative reverse transcription kit (Takara) according to the manufacturer's protocol. The primers for the studied gene (Table S5 ) were added to sample cDNA and a real-time quantitative polymerase chain reaction (PCR) instrument (QuantStudio 1, ThermoFisher Scientific) was used with 40 amplification cycles. Normal cornea was defined as a reference for comparison. The relative fold change of gene expression was analyzed using the 2−ΔΔCt method [10 ,16 ].
Quantitative reverse transcription kit
Manufactured by Takara Bio
The Quantitative reverse transcription kit is a laboratory tool used for the conversion of RNA into complementary DNA (cDNA). It provides the necessary reagents and enzymes to perform this process in a quantitative manner, allowing for accurate measurement of gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Kz 3 f, by Wuhan Servicebio Technology (1 mentions)
Quantstudio 1, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Takara Bio (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
2 protocols using quantitative reverse transcription kit
1
Differentiation of Rabbit Corneal rASCs
2
Gene Expression Profiling of 3D Cultured Cells
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After inoculating on 2D TCP, 3D GelMA, and 100G in SCM and SFM, respectively, for 2 weeks, cells were lysed by Trizol (Invitrogen) to extract RNA. The concentrations of RNA were measured using a Nanodrop 2000 (Thermo scientific). The same amount of RNA was transcribed to cDNA by using a Quantitative Reverse Transcription kit (Takara) according to the manufacturer’s protocol. TaqMan gene expression master mix (Takara) and the primers for the studied gene (Supplementary, Table 1 ) were added to sample cDNA (2 μL), and a StepOnePlus real time PCR system (Applied Biosystems) instrument was used with 40 amplification cycles. Cells cultured on 2D TCP were used as a reference for comparison. β-actin was utilized as an endogenous control for the normalization of studied genes expression levels using the delta delta Ct (ΔΔCt) method. Calculation of 2−ΔΔ Ct was performed to give a comparative fold change of gene expression level relative to actin. Three replicates were measured for each sample to calculate the mean and standard deviation.
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