TRIzol reagent (Invitrogen) or mirVana PARIS System (Ambion, Texas, USA) were respectively employed to isolate total or serum RNA. Subsequently, RNA quantitation and quality determination were performed on the Nanodrop 2000 spectrophotometer platform (Thermo Fisher Scientific, United States). Thereafter, TaqMan miRNA (miR-375 and U6 snRNA) probes (Applied Biosystems, USA) were utilized to perform RT-qPCR according to the manufacturer’s protocol (26 (link)) for mature microRNAs or mRNA using the SYBR Green (Applied Biosystems) method. RT-qPCR was carried out on the 7900 detection system (Applied Biosystems), with U6 small nuclear RNA (snRNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as normalization standards for miRNA and mRNA, respectively. A comparative threshold cycle method was used and values were computed using the 2−ΔΔCt equation. Each reaction in this experiment was run three separate times. The qRT-PCR primer sequences are listed in Table 1 .
Mirvana paris system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MirVana PARIS System is a lab equipment product designed for the isolation and purification of RNA, including small RNAs such as miRNA, from a variety of sample types. It utilizes a unique method to effectively capture and isolate RNA, including small RNA species, while removing DNA, proteins, and other contaminants.
Automatically generated - may contain errors
2 protocols using mirvana paris system
1
Quantitative Analysis of miRNA Expression
2
Quantifying SOX2 Knockdown Efficiency
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Successful knock down of SOX2 by siRNA was assessed by measuring relative mRNA and protein levels with qRT-PCR and Western blot respectively. Briefly, total RNA and protein samples were isolated and purified from the cells using the miRVANA® PARIS system (Ambion, USA). MRNA expression was determined using the the TaqMan® qRT-PCR system (Applied Biosystems, USA) as per manufacturer’s protocols. Relative quantification with the 2-ΔΔCt method, as summarised by Livak and Schmittgen [113 (link)], was used to compare mRNA expression in the functional samples compared to the negative controls. Primer/probe pairs were used to measure the expression of SOX2 (Hs00602736_s1, Applied Biosystems, USA) and an endogenous normalisation control, GAPDH (4331182, Applied Biosystems, USA). The RNA samples were used for down-stream gene and miRNA analysis. Western blot was performed with standard methods, using the enhanced chemiluminescence developed previously by Haan and Behrmann [114 (link)]
. For probing, primary antibodies were used against SOX2 (ab75485, AbCam, UK) and the endogenous normalisation control, GAPDH (ab8245, AbCam, UK).
. For probing, primary antibodies were used against SOX2 (ab75485, AbCam, UK) and the endogenous normalisation control, GAPDH (ab8245, AbCam, UK).
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