RNA Extraction Protocol

From Protists

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Revision as of 16:47, 6 March 2009

Contents 1 Things to Remember 2 Get Cells From Culture 3 Use the RNeasy Plant Mini Kit 4 Icing RNA samples 5 Determine Concentration of RNA 6 RNA Extraction Information Table

Things to Remember

1. Use open (total RNA) RNeasy Plant Mini Kit (Qiagen, available in CSS in 20 or 50rxn packages); back up reagents in back up kit drawers
2. Always use new gloves and change them frequently
3. Sterilize the working area with RNase Zap wipes (wipe once with wipe and then use RO water from bottles to rinse with brown paper towels, and repeat another RO water rinse and towel wipe)…if anything gets touched with non-RNase sterilized gloves/hands/sleeves/etc., then this process must be repeated for those contaminated surfaces throughout the protocol

• • PCR bench area & hood
  • tabletop minicentrifuges, microcentrifuges, dry baths
  • pipetmen & pipet tip barrier tip boxes
  • plastic eppendorf tube holders
  • waste bottle
  • 70% molecular grade ethanol (in orange-capped media bottle in flammables cabinet)
  • Beta-mercaptoethanol (B-ME; in flammables cabinet)
  • pens (sharpies and pens for notes)

1. RNase-free prelabeled tubes (~4-5/reaction) and RNase-free H2O (check that enough in bottle in the kit)
2. transfer supernatant to a fresh tube (avoid aspiration of the interlayer or organic phase)
3. Get ice!
4. ’’If cDNA synthesis directly after:’’

• • water bath to 42C (RT reaction)
  • dry bath to 65C (RT reaction)

Get Cells From Culture

• Follow appropriate protocol for organism or use as fresh & uncontaminated a spun-down cell tube as possible for best yields.
• If cells spun down day prior to total RNA extraction protocol, store overnight in -20C and put in 4C walk in at least 1hr prior to starting protocol.
• If cells frozen in -80C, then thaw overnight in 4C walk in or at RT for at least 1-2hrs prior to starting total RNA extraction protocol.

Use the RNeasy Plant Mini Kit

For reference, follow the kit protocol instructions entitled ‘’purification of total RNA from Animal Cells using Spin Tech’’

1. In a 1.5mL RNase-free tube, add 10uL of B-ME to 990uL of Buffer RLT (in kit), ‘’’all B-ME steps in the hood’’’…calculate approximately how much of this you will need based on your approximate cell weight in your tubes and the number of reactions you are performing (see next step)
2. Disrupt cells by adding B-ME & RLT buffer you made (in hood); use same tip to pipet up and down and then use to add cell + buffer mix to ‘’’Purple’’’ column (step 3)

Number of Cells	Volume of RLT
’’’<5 x 10E6’’’	’’’350uL’’’; will usually be this
1 x 10E7	600uL

1. Homogenize the lysate:

• • Pipet the lysate directly into a prelabeled ‘’’Purple’’’ Qiashredder spin column
  • Centrifuge for ‘’’2min’’’ at ‘’’13,000rpm’’’

1. Remove the spin column, ‘’’add 1 volume 70% EtOH’’’ directly to lysate

• • 1 volume = amount B-ME + RLT Buffer added in step 2, ‘’’most likely 350uL’’’
  • ’’’precipitates (ppt) may be visible; don’t worry – have no effect’’’
  • use same tip to pipet up and down and then use to add to ‘’’Pink’’’ column (step 5)

1. Transfer ‘’’700uL of sample’’’, including ppt to a ‘’’Pink’’’ RNeasy spin column

• • Centrifuge for ‘’’30s’’’ at ‘’’10,000rpm’’’
  • If volume exceeds 700uL, then discard flow-through and repeat

1. Add ‘’’700uL buffer RW1’’’ to spin column

• • Centrifuge for ‘’’30s’’’ at 10,000rpm
  • Carefully remove spin column so flow-through does not touch

1. Add ‘’’500uL buffer RPE’’’ to spin column

• • Centrifuge for ‘’’30s’’’ at 10,000rpm
  • Carefully remove spin column so flow-through does not touch

1. Place spin column into new 1.5mL collection tube from kit
2. Add ‘’’500uL buffer RPE’’’ to spin column

• • Centrifuge for ‘’’2min’’’ at 10,000rpm
  • Carefully remove spin column so flow-through does not touch

1. Place spin column into prelabeled 1.5mL RNase-free eppendorf tube

• • Tube should be labeled with 3code, ‘’’RNA1’’’, date and your initials (top & side)
  • Add ‘’’30uL RNase-free water’’’ from kit directly onto membrane (without touching tip to membrane!)…have extra non-labeled tubes on hand in case lids break off during centrifugation
  • Centrifuge for ‘’’1min’’’ at 10,000rpm
  • ’’’Immediately put tube on ice after spin!!!’’’

1. Repeat step 9 with a new prelabeled 1.5mL RNase-free eppendorf tube

• • Tube should be labeled with 3code, ‘’’RNA2’’’, date and your initials (top & side)
  • ’’’Immediately put tube on ice after spin!!!’’’

Icing RNA samples

• RNA degrades easily, so not putting them on ice will rapidly decrease your yield
• Samples can remain on ice for ~30min
• In finished or not using further, then put labeled RNA tubes in RNA box in -80C freezer

Determine Concentration of RNA

• Follow Nanodrop instructions
• Change nucleic acid type from DNA to ‘’’RNA-40’’’!!!
• Use RNase-free water next to ND-1000 as blank
• Load 2uL of each sample onto Nanodrop
• Take note of curves and OD260/OD280 value; ‘’A value of ‘’’>1.8’’’ is very good quality’’
• In finished or not using further, then put labeled RNA tubes in RNA box in -80C freezer or continue on with cDNA Synthesis protocol

RNA Extraction Information Table

’’’Organism Name’’’	’’’3code’’’	’’’Tube Info’’’ (Person, Date, Weight, etc.)	’’’Cell Comments’’’ (Cell Info: Log/Contaminants/Color?)	’’’RNA Nanodrop Comments’’’ (curve, amount, quality)	’’’Other Notes’’’