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RNA Extraction Protocol (from Nick D. 9/10/2014)

Source of worms: NGM, NAMM

  1. Wash NGM or NAMM plates with M9 and pellet worms in Eppendorf tubes.  Discard supernatant leaving about 100 microliters of M9. You can snap-freeze the eppendorfs in liquid nitrogen and store them at -80C at this stage.
  2. Add 1ml of Trizol (stored in 4 degrees C) to each sample.  Rapidly thaw and freeze the eppendorfs containing Trizol in liquid nitrogen (or -70 degrees C Isopropanol+Dry Ice bath) at least 3x. This ensures adequate worm lysis.
  3. Vortex the eppendorfs at room temperature for 30 minutes.
  4. Spin the worms at max speed at 4 degrees C for 5 minutes. Decant the clear supernatant into tubes from a fresh bag (autoclaving is insufficient to inactivate RNAse) without disturbing the pellet.
  5. Add 200 microliters of chloroform (in the fume hood) and close the eppendorfs firmly.  Vortex the eppendorfs for 10 minutes at room temperature.  This allows for mixing of the Trizol and chloroform layers.
  6. Spin the eppendorfs at max speed at 4C for 10 minutes to separate the aqueous and organic layers.
  7. Separate the colorless and transparent aqueous layer to fresh tubes.  Add in this order 6 microliters of glycoblue (stored in -20C, bottom drawer) then either (500 microliters of isopropanol) OR (150 microliters of 3M sodium acetate pH 5.5 and 1 ml of absolute ethanol). Vortex briefly and place the eppendorfs at -20C overnight (or even over the weekend).
  8. Spin the eppendorfs at max speed at 4C for 25 minutes, after checking that no one else needs that centrifuge.
  9. After the spin you will see a soft blue precipitate. Discard the supernatant completely without disturbing the pellet.  Add 100 microliters of 70% ethanol. 
  10. Spin the tube at max speed at 4C for 10 minutes.
  11. Discard the supernatant and try to remove the alcohol without disturbing the pellet, usually by pipetting.  Air-dry for 10 minutes (not more, or the pellet will be too difficult to resuspend) 

ON COLUMN DNASE DIGESTION

  1. Resuspend pellet in 100 microliters of RNAse free water (Pipet up and down just enough to dislodge thre pellet, then vortex for a few minutes)
  2. From the RNA kit (we use QIAGEN RNeasy mini kit), 350 microliters of Buffer RW1 to an RNA column and centrifuge at 10k rpm for 15 seconds.
  3. Add 350 microliters of Buffer RLT to the RNA in water and mix thoroughly.
  4. Add 250 microliters of 100% ethanol to the RNA mixture and mix thoroughly.  Add mixture to RNA column.  Close gently and centrifuge for 15 seconds at 10k rpm.
  5. Make solution of 70 microliters of water, 2 microliters DNAse, and 8 microliters of DNAse buffer and add to column.  Incubate at 37C for 30 minutes.
  6. Add 350 microliters of RW1 to column and spin for 15 sec @ 10k rpm.
  7. Pipet 500 microliters of Buffer RPE onto spin column.  Close lid and spin for 15 sec @ 10k rpm.
  8. Add 500 microliters of 80% ethanol to spin column. Close the lid and spin for 2 minutes at 10k rpm.
  9. Transfer column to new collection tube and spin for 5 min at full speed with the lid open to dry the membrane.
  10. Elute RNA using RNAse free water (14-24 microliters is sufficient.)
  11. Nanodrop the RNA to determine concentration. 

EPPENDORF DNASE DIGESTION

  1. Resuspend pellet in 100 microliters of RNAse free water (Pipet up and down just enough to dislodge thre pellet, then vortex for a few minutes)
  2. For each RNA sample in 100 microliters of water, add 2 microliters of DNase and 10 microliters of DNase buffer.  Incubate at 37 degrees C for at least 30 minutes.
  3. After incubation, add 350 microliters of Buffer RLT to each RNA sample and mix well.
  4. Add 250 microliters of 100% ethanol and mix well
  5. Add total volume of RNA+ RLT + EtOH (~750 microliters) to RNA spin column, centrifuge at 10,000 RPM for 30 seconds
  6. Throw away eluent from column, add 500 microliters of Buffer RPE to column, centrifuge at 10,000 RPM for 30 seconds.
  7. Throw away eluent from column, add 500 microliters of Buffer RPE to column, centrifuge at 10,000 RPM for 2 minutes
  8. Throw away eluent.  Put column in new collection tube.  Spin column for 1 minute at full speed of the centrifuge.
  9. Put column into microcentrifuge tube.  Add 14-25 microliters of RNase free water to column and let sit for at least 1 minute.  Centrifuge for 2 minutes at max centrifuge speed to elute RNA in water.
  10. Determine RNA concentration (Nanodrop or other mechanism)

EPPENDORF DNASE DIGESTION without columns - we expect higher yield but lower purity

  1. Resuspend pellet in 50 microliters of RNAse free water (Pipet up and down just enough to dislodge thre pellet, then vortex for a few minutes)
  2. For each RNA sample in 50 microliters of water, add 1 microliters of DNase and 5 microliters of DNase buffer.  Incubate at 37 degrees C for at least 30 minutes.
  3. Add 5 microliters of DNAse inactivating reagent, wait 5 minutes, and spin according to kit instructions
  4. Pipet RNA into fresh tube.
  5. Determine RNA concentration (Nanodrop or other mechanism).

cDNA SYNTHESIS (see detailed protocol)

  1. Use cDNA synthesis kit to make cDNAs.  Most kits use 10 microliters of RNA + water (I go for 100 ng of RNA per reaction but have gotten results from 20 ng -- kit maximum is 2000ng per rxn) and 10 microliters of the reverse transcriptase reaction mix. Run reaction at 37C for 60-120 minutes and then 95C for 5 minutes (or whatever your particular RT kit tells you to do).  You can then store at -20 degrees.

REAGENTS WE USE/YOU WILL NEED

  • TRIzol reagent- Ambion/Life Technologies. Stored in 4C. Can purchase in Biochem Stockroom I believe
  • Chloroform- stored in flammables cabinet under hood
  • GlycoBlue –Ambion.  Not sure anyone on campus sells it. Stored in -20C bottom drawer RNA box
  • Turbo DNase – Ambion, and its buffer  Not sure if it sold on campus. Stored in -20C bottom drawer RNA box
  • optional: DNAse inactivating reagent. Ask Nick about where it is stored. Sold in TCSC.
  • RNeasy kit- Qiagen.  Sold in biochem stockroom.  OR Macherey-Nagel RNA cleanup kit.  Columns sold in stockroom but I am not sure about buffers
  • We use the High Capacity cDNA Reverse Transcription Kits from Applied Biosystems.  Can buy these at the Biochem Stockroom.

NOTES

  1. Clean the pipettors with RNAse-away
  2. Use a fresh box of tips or filter tips to avoid RNAse
  3. Wear a fresh pair of gloves to avoid RNAse and to keep from getting TRIzol on your skin
  4. Wear safety goggles to keep from getting TRIzol in your eye
  5. Wear the cleanest lab coat you can find to keep from getting TRIzol on your skin and to announce to everyone that you're working with RNA.

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