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

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==Safety Information==
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* [[SOP-_Phenol|SOP - Phenol]]
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* [[SOP_-_Chloroform|SOP - Chloroform]]
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==Materials==
 
==Materials==
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*RNeasy Kit (Invitrogen)
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*PureLink RNA Mini Kit (Invitrogen cat#12183-018A)
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*Mouse Tissue (20-30 mg, about a 3mm cube)
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*Mouse Tissue (50-100 mg, about a 3mm cube)
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*Label tubes, for each sample need 2 eppies, one RNeasy
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*TRIZol (Invitrogen cat# 12183-555)
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*Chloroform (in solvent cabinet)
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*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)
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*70% Ethanol make with RNAase free water and 100% Ethanol.
  
 
==Protocol==
 
==Protocol==
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#Cut tissue and weigh in a fresh tube to ensure a sample of up to 30 mg tissue
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#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.
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#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
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#Add 1 mL TRIzol reagent to each 2 mL tube.
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#Using dounce homogenizer, homogenize tissue 10x and transfer to a clean tube
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#Add 1 ball bearing to each tube.
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#Centrifuge 3 min at room temperature at 14 000 RPM
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#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.
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#Remove centrifuge to a clean tube
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#Incubate 5 minutes at room temperature.
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#Add 600 uL of 70% ethanol and mix immediately by pipetting
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#Transfer sample to a new 1.5 vial. The ball bearings are reusable after cleaning so do not discard them.
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#Remove 700 uL of mixture (a precipitate may have formed) and add to a RNeasy spin column in a collection tube
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#Add 200 uL Chloroform and shake vigourously by hand for 15 seconds.  Do NOT vortex.
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#Spin 15s at 14 000 RPM.  Discard flow through
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#Incubate at room temperature for 2-3 minutes.
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#Add 700 uL RW1 to spin colum
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#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.
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#Per sample, combine 10 uL DNAse I (in Enzymes Box) with 70 uL RDD (in Molecular Biology Box) and mix
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#Add 400 uL of 70% ethanol to a fresh tube.
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#Add this mixture (80 uL per sample) to spin columns and sit on bench for 15 min
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#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.
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#Add 350 uL RW1 to spin column
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#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.
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#Spin 15s at 14 000 RPM.  Discard flow through
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#Spin 15 seconds on max.  Discard flow through.  Add remaining sample, respin and discard flow through.
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#Add 500 uL RPE
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#Add 700 uL Wash Buffer I to spin column.
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#Spin 2 min at 14 000 RPM
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#Spin 15 seconds on max.  Discard flow through and collection tube, add a new collection tube.
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#Remove spin column to a new eppie and spin again to remove residual RPE
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#Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
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#Add 50 uL RNAse free water and spin 1 min to elute RNA
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#Spin 15 seconds on max.  Discard the flow through and keep the same collection tube.
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#Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
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#Spin 15 seconds on max.  Discard the flow through and keep the same collection tube.
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#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.
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#Incubate at room temperature for 1 minute.
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#Spin 2 minutes at 12500 rpm to get purified RNA.
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#Quantify the RNA using the nanodrop.
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[[Category:qPCR]]
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[[Category:Expression]]
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[[Category:Transcription]]
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[[Category:RNA]]
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[[Category:Mouse Work]]
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[[Category:Tissues]]

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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