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

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(added categories)
(updated for purelink kits.)
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==Materials==
 
==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==
 
==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.
  
  

Revision as of 16:51, 11 January 2012

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.


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 dry ice (if frozen).
  2. Add 1 mL TRIzol reagent to each tube.
  3. Using tissue grinder, homogenize tissue for ~30s until homogeneous (no clumps).
  4. Incubate 5 minutes at room temperature.
  5. Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex.
  6. Incubate at room temperature for 2-3 minutes.
  7. 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.
  8. Transfer 600 uL of the upper phase to a fresh tube.
  9. Add 600 uL of 70% ethanol and mix by vortexing.
  10. Remove 700 uL of mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
  11. Spin 15s on max (press button). Discard flow through. Add remaining sample and respin.
  12. Add 700 uL Wash Buffer I to spin column.
  13. Spin 15s on max. Discard flow through and the collection tube. Get a new collection tube.
  14. Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
  15. Spin 15s on max. Discard the flow through and replace the collection tube.
  16. Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
  17. Spin 15s on max. Discard the flow through and keep the same collection tube.
  18. 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.
  19. Incubate at room temperature for 1min.
  20. Spin 2 min on max to get purified RNA.
  21. Quantify the RNA using the nanodrop.
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