Changes

*RNeasy PureLink RNA Mini Kit (Invitrogencat#12183-018A)*Mouse Tissue (2050-30 100 mg, about a 3mm cube)*TRIZol (Invitrogen cat# 12183-555)*Chloroform (in solvent cabinet)*Label tubes, for each sample need : 2 eppies.0 mL tube (to cut sample into), one RNeasy1.5 mL tubes (X2), 1 spin column*, 1 recovery tube*, 1 collection tube* (*= in the PureLink kit)*70% Ethanol make with RNAase free water and 100% Ethanol.

#Cut ~50-100 mg of tissue and weigh . If tissue is in RNAlater, cut at room temperature. If tissue is frozen, cut on dry ice. Weigh into a fresh 2mL eppendorf tube to ensure a sample of up to 30 mg tissueand keep on ice.#Prepare buffer RLT by adding 10 uL B-ME per Add 1 mL of RLT in a clean 15 TRIzol reagent to each 2 mL falcon tube. Need 600 uL per sample#Add 1 ball bearing to each tube.#Using dounce homogenizertissue grinder, homogenize tissue 10x and transfer for 3 minutes at 25 Hz (make sure there are no remaining clumps, maybe take longer for muscle). If chunks remain after 3 minutes, homogenize until chunks are gone in 2 minute increments.#Incubate 5 minutes at room temperature.#Transfer sample to a clean tubenew 1.5 vial. The ball bearings are reusable after cleaning so do not discard them.#Centrifuge 3 min Add 200 uL Chloroform and shake vigourously by hand for 15 seconds. Do NOT vortex.#Incubate at room temperature at 14 000 RPMfor 2-3 minutes.#Remove Centrifuge in a cold eppendorf centrifuge to (4C) on maximum for 15 minutes. Sample will separate into a clean tubelower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA. (WAT tissue may separate into 3 phases, RNA is the top, colorless phase. #Add 600 400 uL of 70% ethanol to a fresh tube.#Carefully transfer 400 uL of the upper phase to the ethanol tube and mix immediately by pipettingvortexing. The remaining chloroform in the previous vial should be disposed of in the phenol waste container under the hood.#Remove 700 uL of ethanol/upper phase mixture (a precipitate may have formedinvert briefly to mix before taking sample) and add to a RNeasy spin column in a collection tube.#Spin 15s at 14 000 RPM15 seconds on max. Discard flow through. Add remaining sample, respin and discard flow through.#Add 700 uL RW1 Wash Buffer I to spin columcolumn.#Per sample, combine 10 uL DNAse I (in Enzymes Box) with 70 uL RDD (in Molecular Biology Box) Spin 15 seconds on max. Discard flow through and mixcollection tube, add a new collection tube. #Add this mixture (80 500 uL per sampleWash Buffer II (make sure ethanol was added to the wash buffer) to the spin columns and sit on bench for 15 min#Add 350 uL RW1 to spin columncartridge.#Spin 15s at 14 000 RPM15 seconds on max. Discard the flow throughand keep the same collection tube.#Add Repeat wash by adding 500 uL RPEWash Buffer II to the spin cartridge.#Spin 2 min at 14 000 RPM15 seconds on max. Discard the flow through and keep the same collection tube.#Remove spin column Spin 1 minute on max to a new eppie dry the cartridge. Discard the collection tube and place spin again to remove residual RPE#column into a clean recovery tube. Add 50 100 uL RNAse RNAase free water to the center of each tube. This can be adjusted to between 30 and spin 300 uL elution buffer if necessary. #Incubate at room temperature for 1 min minute.#Spin 2 minutes at 12500 rpm to elute get purified RNA.#Quantify the RNA using the nanodrop.  [[Category:qPCR]][[Category:Expression]][[Category:Transcription]][[Category:RNA]][[Category:Mouse Work]][[Category:Tissues]]