QuantiFluor® RNA System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 200µl of QuantiFluor® RNA Dye working solution to the sample. Incubate for 5 minutes at room temperature, protected from light	Measure fluorescence at 492nmEx/540nmEm

Protocol tips
Add 200µl of QuantiFluor® RNA Dye working solution to the sample. Incubate for 5 minutes at room temperature, protected from light
Downstream tips
Measure fluorescence at 492nmEx/540nmEm

Publication protocol

Using Plasma/Serum Circulating and Exosomal RNA Purification Kit (51000, Norgen Biotek Corp., ON, Canada), total RNA was extracted from the purified exosomes that were obtained from the same amount of the plasma. 5 pg of celmiR-39 mimic (Applied Biosystems, CA, USA) was added as an external control. RNA was subjected to complementary DNA (cDNA) synthesis using a TaqMan MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems, CA, USA) and PCR was performed using TaqMan Fast Advanced Master Mix and StepOnePlus realtime PCR system (both from Thermo Fisher Scientific, MA, USA). The data of each miRNA was analysed by the delta-delta Ct method using data of celmiR-39 and normalised to the amount of applied RNA. The amount of total RNA was determined using QuantiFluor RNA system (Promega, WI, USA). For detection of cellular RNA, miRNeasy kit (217004, Qiagen, Venlo, Netherlands) was used in the extraction step. MiRNA was then analysed with the same method as described above, and the data was normalised to the expression of U6 snRNA. For analysis of mRNA or primary miRNA, extracted RNA was subjected to cDNA synthesis using PrimeScript RT Master Mix (TAKARA, Shiga, Japan) and PCR was performed using TaqMan Fast Advanced Master Mix and StepOnePlus realtime PCR system. The data was normalised to the expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase). These primers were used: TaqMan MicroRNA Assays of hsa-let-7i-5p, hsa-miR-19b-3p, hsa-miR-25-3p, hsa-miR-92a-3p and U6 snRNA; TaqMan Pri-miRNA Assay of has-let-7i; and TaqMan Gene Expression Assay of GAPDH (Applied Biosystems, CA, USA

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Manufacturer protocol

Download the product protocol from Promega for QuantiFluor® RNA System below.

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