Preparation of RNA Samples from Mouse Tissues: Difference between revisions
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. | ||
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#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. | ||