Snap-frozen tissues were first treated with RNAlater™-ICE Frozen Tissue Transition Solution (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions, to allow handling of tissue without RNA degradation occurring due to thawing of sample. Tissue sections were placed in 1 mL TRI Reagent® Solution (ThermoFisher, Waltham, MA, USA) supplemented with the addition of 10 mM MgCl2 to aid recovery of microRNAs (Kim et al. 2012 (link)). Samples were completely homogenised in a bead mill (Retsch Technology GmbH, Haan, Germany) at a frequency of 30 cycles per second for 15 min. Phase separation was carried out using chloroform. Total RNA was precipitated from the aqueous phase by means of an overnight incubation at − 20 °C with isopropanol. A total of 1.2 μl Invitrogen™ GlycoBlue™ Coprecipitant (ThermoFisher, Waltham, MA, USA) was added prior to incubation to aid pellet recovery. RNA pellets were ethanol-washed twice and re-suspended in 1× TE buffer, pH 8.0. RNA quality and concentration were assessed by NanoDrop spectrophotometry (NanoDrop, Wilmington, DE, USA).
Invitrogen glycoblue coprecipitant
Manufactured by Thermo Fisher Scientific
Sourced in United States
Invitrogen™ GlycoBlue™ Coprecipitant is a reagent used for the precipitation and visualization of nucleic acids in molecular biology applications. It functions by forming a colored precipitate with nucleic acids, allowing for easy detection and recovery.
Automatically generated - may contain errors
2 protocols using invitrogen glycoblue coprecipitant
1
Extraction of High-Quality RNA from Snap-Frozen Tissues
2
Robust Total RNA Extraction from Frozen Tissues
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Snap-frozen tissues were first treated with RNAlater™-ICE Frozen Tissue Transition Solution (ThermoFisher, Waltham, MA, USA) according to the manufacturer's instructions, in order to allow handling of the tissue without RNA degradation occurring due to thawing of sample. Tissue sections were then placed in 1 mL TRI Reagent ® Solution (ThermoFisher, Waltham, MA, USA) supplemented with the addition of 10mM MgCl2 to aid recovery of microRNAs (Kim and others 2012) . Samples were then completely homogenized in a bead mill (Retsch Technology GmbH, Haan, Germany) at a frequency of 30 cycles per second for 15 mins. Phase separation was carried out using chloroform.
Total RNA was precipitated from the aqueous phase by means of an overnight incubation at -20°C with isopropanol. 1.2µl Invitrogen™ GlycoBlue™ Coprecipitant (ThermoFisher, Waltham, MA, USA) was added prior to incubation to aid pellet recovery. RNA pellets were then ethanol-washed twice and resuspended in 1X TE buffer, pH8.0. RNA quality and concentration were assessed by NanoDrop spectrophotometry (NanoDrop, Wilmington, DE, USA).
Total RNA was precipitated from the aqueous phase by means of an overnight incubation at -20°C with isopropanol. 1.2µl Invitrogen™ GlycoBlue™ Coprecipitant (ThermoFisher, Waltham, MA, USA) was added prior to incubation to aid pellet recovery. RNA pellets were then ethanol-washed twice and resuspended in 1X TE buffer, pH8.0. RNA quality and concentration were assessed by NanoDrop spectrophotometry (NanoDrop, Wilmington, DE, USA).
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