Difference between revisions of "Preparation of RNA Samples from Mouse Tissues"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (changed volume to remove of upper phase (typo from original protocol)) |
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#Add 1 mL TRIzol reagent to each 2 mL tube. | #Add 1 mL TRIzol reagent to each 2 mL tube. | ||
#Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on ice. | #Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on ice. | ||
| − | #Using tissue grinder, homogenize tissue for | + | #Using tissue grinder, homogenize tissue for 3 minutes at 25Hz (make sure there are no remaining clumps). |
#Incubate 5 minutes at room temperature. | #Incubate 5 minutes at room temperature. | ||
#Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex. | #Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex. | ||
#Incubate at room temperature for 2-3 minutes. | #Incubate at room temperature for 2-3 minutes. | ||
#Centrifuge in a cold eppendorf centrifuge on maximum for 15 minutes. Sample will separate into a lower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA. | #Centrifuge in a cold eppendorf centrifuge on maximum for 15 minutes. Sample will separate into a lower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA. | ||
| − | # | + | #Add 400 uL of 70% ethanol to a fresh tube. |
| − | # | + | #Transfer 400 uL of the upper phase to the ethanol tube and mix by vortexing. |
#Remove 700 uL of mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube. | #Remove 700 uL of mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube. | ||
| − | #Spin 15s on max | + | #Spin 15s on max. Discard flow through. Add remaining sample and respin. |
#Add 700 uL Wash Buffer I to spin column. | #Add 700 uL Wash Buffer I to spin column. | ||
| − | #Spin 15s on max. Discard flow through and the collection tube. Get a new collection tube. | + | #Spin 15s on max. Discard flow through and the collection tube. <s>Get a new collection tube.</s> |
#Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge. | #Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge. | ||
#Spin 15s on max. Discard the flow through and replace the collection tube. | #Spin 15s on max. Discard the flow through and replace the collection tube. | ||
| Line 27: | Line 27: | ||
#Spin 1 min on max to dry the cartridge. Discard the collection tube and place into a clean recovery tube. Add 100 uL RNAase free water to the center of each tube. This can be adjusted to between 30 and 300 uL elution buffer if necessary. | #Spin 1 min on max to dry the cartridge. Discard the collection tube and place into a clean recovery tube. Add 100 uL RNAase free water to the center of each tube. This can be adjusted to between 30 and 300 uL elution buffer if necessary. | ||
#Incubate at room temperature for 1min. | #Incubate at room temperature for 1min. | ||
| − | #Spin 2 min | + | #Spin 2 min 12500 rpm to get purified RNA. |
#Quantify the RNA using the nanodrop. | #Quantify the RNA using the nanodrop. | ||
Revision as of 18:13, 28 January 2016
Materials
- PureLink RNA Mini Kit (Invitrogen cat#12183-018A)
- Mouse Tissue (50-100 mg, about a 3mm cube)
- TRIZol (Invitrogen cat# 12183-555)
- Chloroform (in solvent cabinet)
- Label tubes, for each sample need 3 eppies, at least one of which is 2 mL and one of which is a recovery tube from the PureLink kit.
- 70% Ethanol make with RNAase free water and 100% Ethanol.
Protocol
- Add 1 mL TRIzol reagent to each 2 mL tube.
- Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on ice.
- Using tissue grinder, homogenize tissue for 3 minutes at 25Hz (make sure there are no remaining clumps).
- Incubate 5 minutes at room temperature.
- Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex.
- Incubate at room temperature for 2-3 minutes.
- Centrifuge in a cold eppendorf centrifuge on maximum for 15 minutes. Sample will separate into a lower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA.
- Add 400 uL of 70% ethanol to a fresh tube.
- Transfer 400 uL of the upper phase to the ethanol tube and mix by vortexing.
- Remove 700 uL of mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
- Spin 15s on max. Discard flow through. Add remaining sample and respin.
- Add 700 uL Wash Buffer I to spin column.
- Spin 15s on max. Discard flow through and the collection tube.
Get a new collection tube. - Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
- Spin 15s on max. Discard the flow through and replace the collection tube.
- Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
- Spin 15s on max. Discard the flow through and keep the same collection tube.
- Spin 1 min on max to dry the cartridge. Discard the collection tube and place into a clean recovery tube. Add 100 uL RNAase free water to the center of each tube. This can be adjusted to between 30 and 300 uL elution buffer if necessary.
- Incubate at room temperature for 1min.
- Spin 2 min 12500 rpm to get purified RNA.
- Quantify the RNA using the nanodrop.
