Preparation of RNA Samples from Mouse Tissues: Difference between revisions

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 ==Materials==
-*RNeasy Kit (Invitrogen)
+*PureLink RNA Mini Kit (Invitrogen cat#12183-018A)
-*Mouse Tissue (20-30 mg, about a 3mm cube)
+*Mouse Tissue (50-100 mg, about a 3mm cube)
-*Label tubes, for each sample need 2 eppies, one RNeasy
+*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.
 ==Protocol==
-#Cut tissue and weigh in a fresh tube to ensure a sample of up to 30 mg tissue
+#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 dry ice (if frozen).
-#Prepare buffer RLT by adding 10 uL B-ME per mL of RLT in a clean 15 mL falcon tube.  Need 600 uL per sample
+#Add 1 mL TRIzol reagent to each tube.
-#Using dounce homogenizer, homogenize tissue 10x and transfer to a clean tube
+#Using tissue grinder, homogenize tissue for ~30s until homogeneous (no clumps).
-#Centrifuge 3 min at room temperature at 14 000 RPM
+#Incubate 5 minutes at room temperature.
-#Remove centrifuge to a clean tube
+#Add 200 uL Chloroform and shake vigourously by hand for 15s.  Do not vortex.
-#Add 600 uL of 70% ethanol and mix immediately by pipetting
+#Incubate at room temperature for 2-3 minutes.
-#Remove 700 uL of mixture (a precipitate may have formed) and add to a RNeasy spin column in a collection tube
+#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.
-#Spin 15s at 14 000 RPM.  Discard flow through
+#Transfer 600 uL of the upper phase to a fresh tube.
-#Add 700 uL RW1 to spin colum
+#Add 600 uL of 70% ethanol and mix by vortexing.
-#Per sample, combine 10 uL DNAse I (in Enzymes Box) with 70 uL RDD (in Molecular Biology Box) and mix
+#Remove 700 uL of mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
-#Add this mixture (80 uL per sample) to spin columns and sit on bench for 15 min
+#Spin 15s on max (press button).  Discard flow through.  Add remaining sample and respin.
-#Add 350 uL RW1 to spin column
+#Add 700 uL Wash Buffer I to spin column.
-#Spin 15s at 14 000 RPM.  Discard flow through
+#Spin 15s on max.  Discard flow through and the collection tube.  Get a new collection tube.
-#Add 500 uL RPE
+#Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
-#Spin 2 min at 14 000 RPM
+#Spin 15s on max.  Discard the flow through and replace the collection tube.
-#Remove spin column to a new eppie and spin again to remove residual RPE
+#Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
-#Add 50 uL RNAse free water and spin 1 min to elute RNA
+#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 on max to get purified RNA.
+#Quantify the RNA using the nanodrop.