RNAzol® RT

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- To reduce RNA degradation do not wash cells before the addition of RNAzol® RT. - To reduce DNA contamination, use sufficient amount of RNAzol® RT based on the area of the culture dish and not on cell number.	To reduce DNA contamination extend the incubation time to 15 minutes before sedimentation.

Upstream tips
- To reduce RNA degradation do not wash cells before the addition of RNAzol® RT. - To reduce DNA contamination, use sufficient amount of RNAzol® RT based on the area of the culture dish and not on cell number.
Protocol tips
To reduce DNA contamination extend the incubation time to 15 minutes before sedimentation.

Publication protocol

RNAzol RT (Molecular Research Center, USA) was used to extract total RNA from both cellular and exosomal sources [4]. 1 mL of RNAzol was added to the exosomal pellet and homogenised using a blunt needle syringe. This homogenate is added to 0.4 mL of water and left to incubate on ice for up to 15 min. The sample was then centrifuged at 4 °C, at 12,000g for 15 min. 1 mL of the resulting supernatant was removed and purification performed via the addition of 5 μL of 4-bromoanisole. The sample was incubated for 3–4 min, and centrifuged at 12,000g, 4 °C, for 10 min. The supernatant was removed from the sample, and 1 volume of isopropanol was added with 5 μL of glycogen (25 mg/mL) to precipitate the RNA. This mixture was then incubate on dry ice for 15 min followed by centrifugation at 12,000g, 4 °C, for 10 min. The resulting RNA pellet was then washed twice in 75% ethanol and centrifuge at 8000g for 5 min to collect the pellet. After discarding the supernatant, the exosomal RNA pellet was solubilized with the addition of 50 μL of RNA/DNA free water.

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Manufacturer protocol

Download the product protocol from Molecular Research Center, Inc. for RNAzol® RT below.

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