mirVana™ miRNA Isolation Kit, with phenol

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercapto ethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation. - To capture more amount of mRNA, modify the amount of lysis buffer:ethanol (from 1:1 to 1:1.5) during the binding step.	- Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA. - Digest with 100 μg/mL Proteinase K in the presence of 0.5% SDS at 37°C for 30 min.

Protocol tips
- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercapto ethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation. - To capture more amount of mRNA, modify the amount of lysis buffer:ethanol (from 1:1 to 1:1.5) during the binding step.
Downstream tips
- Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA. - Digest with 100 μg/mL Proteinase K in the presence of 0.5% SDS at 37°C for 30 min.

Publication protocol

For RNA isolation, total RNA was extracted and isolated from tissue samples or cell lines using either the mirVana miRNA isolation kit (Ambion, Austin, TX) or the TRIzol method. Trizol of 1 mL was added and the solution was mixed homogeneously for 10 min. The mixture was then transferred into eppendorf tubes (EP, 1.5 mL) with 200 μL chloroform. After 15 min shake, the EP tubes were centrifuged at 4°C for 15 min (12000 × g). The supernate was transferred into other EP tubes and mixed with isopyknic isopropanol for 15 s. The centrifugation (4°C, 10 min, 12000 × g) was performed again and the supernate was discarded. The precipitate was washed by 75% ethonal twice and dissolved into 30 μL diethypyrocarbonate (DEPC) after drying to obtain RNA stock solution. After isolation, the concentration of RNA was assessed using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA) and the RAN solution was stored at -80°C for further use.

For qRT-PCR, genes were amplified by specific oligonucleotide primer, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. The detection and quantification contained the following steps: first, reverse transcription was performed at 55°C for 30 min, initial activation for 15 min at 95°C, next 40 cycles of denaturation were conducted at 94°C for 15 s, then annealing for 30 s at 55°C, extension for 30 s at 72°C. The expression level was normalized using U6 small nuclear RNA by the 2-ΔCt method. The ΔCt values were normalized to GAPDH level.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for mirVana™ miRNA Isolation Kit, with phenol below.

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