Oct 02, 2024
RNA isolation
- 1University of veterinary medicine, Vienna
Protocol Citation: Angkana Kidtiwong 2024. RNA isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpznpplzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 02, 2024
Last Modified: October 02, 2024
Protocol Integer ID: 108813
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Abstract
RNA isolation for sample stored in TRIzol reagent
RNA isolation
RNA isolation
23m
23m
Sample in TRIzol
Equipment
accuSpin Micro 17R refrigerated centrifuge
NAME
refrigerated microcentrifuge
TYPE
Fisher Scientific
BRAND
Micro17R
SKU
Prepare the centrifuge at 4°C and meanwhile thaw the frozen sample (in TRIzol, stored at -80°C) at room temperature for about 10-15 minutes.
Add chloroform at 20% of the sample volume to the sample tube (typically, 1 ml of TRIzol sample, so add 200 µl of chloroform in sample tube) and incubate at room temperature for 5 minutes. Mixing from time to time during the incubation
Centrifuge at 13,000 rpm, 4°C for 15 minutes.
Equipment
accuSpin Micro 17R refrigerated centrifuge
NAME
refrigerated microcentrifuge
TYPE
Fisher Scientific
BRAND
Micro17R
SKU
In a meanwhile, prepare the mixture of RNA precipitation as follow:
a. Isopropanol: 0.8 times the total volume of supernatant plus 400 µl DEPC water (typically, take 400 µl of supernatant after centrifugation (clear part), add 640 µl of isopropanol) per sample.
b. DEPC water: 400 µl per sample.
c. Glycoblue: 1-2 µl per sample.
- Note: Always prepare 2-3 extra mixtures.
Transfer 400 µl of supernatant after centrifugation (clear part) to a new Eppendorf tube - Carefully pipetting
Add 1,041 µl of the RNA precipitation mixture (400 µl DEPC water + 640 µl isopropanol + 1 µl Glycoblue) to the supernatant. Incubate on ice for 10-30 minutes.
Centrifuge at 13,000 rpm, 4°C for 15 minutes.
Equipment
accuSpin Micro 17R refrigerated centrifuge
NAME
refrigerated microcentrifuge
TYPE
Fisher Scientific
BRAND
Micro17R
SKU
Check if a blue pellet has formed at the bottom of the Eppendorf tube, then discard the supernatant and add 1 ml of 75% ethanol (ETOH volume is proportional to TRIzol volume).
Centrifuge at 13,000 rpm, 4°C for 5 minutes.
Equipment
accuSpin Micro 17R refrigerated centrifuge
NAME
refrigerated microcentrifuge
TYPE
Fisher Scientific
BRAND
Micro17R
SKU
Discard the supernatant, then add 1 ml of 75% ethanol and transfer everything to a new Eppendorf tube – Ensure the pellet is transferred as well.
Centrifuge at 13,000 rpm, 4°C for 5 minutes.
Equipment
accuSpin Micro 17R refrigerated centrifuge
NAME
refrigerated microcentrifuge
TYPE
Fisher Scientific
BRAND
Micro17R
SKU
Discard all ethanol and air-dry the pellet in the chamber for 5 minutes.
Add 20 µl of nuclease-free water to each Eppendorf tube
Prepare the RNA treatment mixture (10 µl per sample) of:
a. 6 µl of nuclease-free water.
b. 3 µl of DNase buffer.
c. 1 µl of DNase enzyme.
Note: Always prepare 1-2 extra mixtures.
Add 10 µl of the RNA treatment mixture to the sample and incubate at 37°C for 20 minutes on thermo shaker incubator
20m
Add 3 µl of DNase inactivation reagent and incubate for 2 minutes at room temperature.
Centrifuge at 13,000 rpm, 4°C for 3 minutes.
Equipment
accuSpin Micro 17R refrigerated centrifuge
NAME
refrigerated microcentrifuge
TYPE
Fisher Scientific
BRAND
Micro17R
SKU
3m
Transfer 25 µl of the supernatant to a 1.5 ml Eppendorf tube – keep on ice until RNA measurement.