Difference between revisions of "Preparation of RNA Samples from Mouse Tissues"

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*TRIZol (Invitrogen cat# 12183-555)
 
*TRIZol (Invitrogen cat# 12183-555)
 
*Chloroform (in solvent cabinet)
 
*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.
+
*Label tubes, for each sample need: 2.0 mL tube (to cut sample into), 1.5 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.
 
*70% Ethanol make with RNAase free water and 100% Ethanol.
  
 
==Protocol==
 
==Protocol==
 +
#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.
 
#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.
+
#Add 1 ball bearing to each tube.
−
#Using tissue grinder, and after adding the ball bearings to every vial, homogenize tissue for 3 min at 25Hz (make sure there are no remaining clumps, maybe take longer for muscle).
+
#Using tissue grinder, homogenize tissue 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.
 
#Incubate 5 minutes at room temperature.
 
#Transfer sample to a new 1.5 vial. The ball bearings are reusable after cleaning so do not discard them.
 
#Transfer sample to a new 1.5 vial. The ball bearings are reusable after cleaning so do not discard them.
−
#Add 200 uL Chloroform and shake vigourously by hand for 15s.  Do not vortex.
+
#Add 200 uL Chloroform and shake vigourously by hand for 15 seconds.  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 (4C) 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 (4C) 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. (WAT tissue may separate into 3 phases, RNA is the top, colorless phase.  
 
#Add 400 uL of 70% ethanol to a fresh tube.
 
#Add 400 uL of 70% ethanol to a fresh tube.
 
#Carefully transfer 400 uL of the upper phase to the ethanol tube and mix by vortexing. The remaining chloroform in the previous vial should be disposed of in the phenol waste container under the hood.
 
#Carefully transfer 400 uL of the upper phase to the ethanol tube and mix by vortexing. The remaining chloroform in the previous vial should be disposed of in the phenol waste container under the hood.
−
#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 ethanol/upper phase 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, respin and discard flow through.
+
#Spin 15 seconds on max.  Discard flow through.  Add remaining sample, respin and discard flow through.
 
#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 the collection tube.  
+
#Spin 15 seconds on max.  Discard flow through and collection tube, add a new collection tube.  
 
#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 15 seconds on max.  Discard the flow through and keep the same collection tube.
 
#Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
 
#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 15 seconds 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.  
+
#Spin 1 minute on max to dry the cartridge.  Discard the collection tube and place spin column 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 1 minute.
−
#Spin 2 min at 12500 rpm to get purified RNA.
+
#Spin 2 minutes at 12500 rpm to get purified RNA.
 
#Quantify the RNA using the nanodrop.
 
#Quantify the RNA using the nanodrop.
  

Latest revision as of 20:51, 22 October 2018

Safety Information

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: 2.0 mL tube (to cut sample into), 1.5 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.

Protocol

  1. 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.
  2. Add 1 mL TRIzol reagent to each 2 mL tube.
  3. Add 1 ball bearing to each tube.
  4. Using tissue grinder, homogenize tissue 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.
  5. Incubate 5 minutes at room temperature.
  6. Transfer sample to a new 1.5 vial. The ball bearings are reusable after cleaning so do not discard them.
  7. Add 200 uL Chloroform and shake vigourously by hand for 15 seconds. Do NOT vortex.
  8. Incubate at room temperature for 2-3 minutes.
  9. Centrifuge in a cold eppendorf centrifuge (4C) 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. (WAT tissue may separate into 3 phases, RNA is the top, colorless phase.
  10. Add 400 uL of 70% ethanol to a fresh tube.
  11. Carefully transfer 400 uL of the upper phase to the ethanol tube and mix by vortexing. The remaining chloroform in the previous vial should be disposed of in the phenol waste container under the hood.
  12. Remove 700 uL of ethanol/upper phase mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
  13. Spin 15 seconds on max. Discard flow through. Add remaining sample, respin and discard flow through.
  14. Add 700 uL Wash Buffer I to spin column.
  15. Spin 15 seconds on max. Discard flow through and collection tube, add a new collection tube.
  16. Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
  17. Spin 15 seconds on max. Discard the flow through and keep the same collection tube.
  18. Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
  19. Spin 15 seconds on max. Discard the flow through and keep the same collection tube.
  20. Spin 1 minute on max to dry the cartridge. Discard the collection tube and place spin column 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.
  21. Incubate at room temperature for 1 minute.
  22. Spin 2 minutes at 12500 rpm to get purified RNA.
  23. Quantify the RNA using the nanodrop.
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