RNA extraction
- Read the SOPs for each of the reagents used in this protocol.
- Read the manufacturer's RNAzol RT protocol for Total RNA Isolation.
- Read this protocol.
- Verify sufficient quantities/availability of reagents/equipment.
- Wear clean gloves and change gloves frequently.
- Aliquot 500uL of RNAzol RT to pestle tubes and store on ice.
- Transfer tissue to pestle tubes containing RNAzol RT.
- Homogenize immediately with disposable pestle.
- Immediately add additional 500uL of RNAzol RT to pestle tube.
- Vortex 15s.
- Add 400uL of 0.1% DEPC-treated H2O.
- Vortex 15s.
- Incubate at room temperature (RT) for 15mins.
- Centrifuge 12,000g for 15mins @ RT.
- Transfer 750uL of supernatant (do not disturb pellet) to sterile 1.7mL snap-cap tube. (Discard remaining liquid in RNAzol RT Hazardous Waste container in fume hood. Leave old tube open in fume hood over night and then discard in regular trash.)
- Add 1 volume of isopropanol.
- Vortex 5s.
- Incubate @ RT for 15mins.
- Centrifuge 12,000g for 10mins @ RT.
- Discard supernatant; do not disturb pellet.
- Add 400uL of 75% ethanol, centrifuge 4,000g for 3mins @ RT.
- Repeat steps 20 and 21 one time.
- Repeat step 20.
- Centrifuge 4,000g for 1min @ RT.
- Removal residual ethanol.
- Immediately resuspend pellet in appropriate volume of 0.1% DEPC-H2O (volume is dependent upon pellet size, but 50uL is usually sufficient).
- Keep sample on ice for short-term storage (i.e. no more than 2hrs) or store @ -80C.
Reverse Transcription
Standard Operating Protocol (SOP)
Written 20150702 by Sam White.
Reagents:
- M-MLV Reverse Transcriptase (Promega: M1701)
- Primers (oligo dT: Promega: C1101 OR random: Promega: C1181)
- 10mM dNTPs (Promega: U1511)
Personal Protective Equipment (PPE):
- Gloves
Equipment:
- Pipettes (10 - 1000uL)
- Filtered pipette tips
- 0.5mL snap-cap microfuge tubes (Genesee: 22-178A)
- Sterile 1.7mL snap-cap microfuge tubes (Genesee: 22-281S)
- Thermal cycler, water bath, or heating block capable of 37C OR 42C.
- vortexer
- ice
Procedure
Total Time: ~ 1.5 - 2.0hrs
Cost/sample: ~ $1.50
IMPORTANT: A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used in this protocol are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.
- Read the manufacturer's protocol.
- Read this protocol.
- Verify sufficient quantities of reagents and samples before beginning.
- Wear clean gloves.
- Thaw all RNA and reagents on ice. Prepare all reactions on ice.
- Transfer 1ug of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O.
- Add 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT Cat#C1101 in this example). Total volume (RNA + primers) should equal 18.25uL.
- Heat samples at 70C for 5 min in thermal cycler, heating block, or water bath.
- Place samples on ice IMMEDIATELY.
- Make Master Mix:Per Reaction
- 5 uL 5x Buffer (M-MLV RT Buffer)
- 1.25 uL 10mM dNTPs
- 0.5 uL M-MLV RT per ug of RNA
- Mix well by flicking; do not vortex.
- Add 6.75uL of master mix to each reaction.
- Mix by pipetting; do not vortex.
- Incubate @ 42C for 1hr for oligo dT primers OR @ 37C for random primers.
- Heat inactivate @ 95C for 3 min.
- Spot spin and store @-20C.
Results
Sucessfully extracted RNA from the tissue samples and checked the RNA concentrations of all samples using the NANO drop (ND-1000) shown below, all RNA extractions were successful.
(note: I thought it was interesting that all of the day time samples had a higher concentration than the night time samples though I can not draw any conclusions from it an interesting find)
CDNA was sucessfull the next step will be to run a QPCR to check expression levels.
RNA Concentrations ng/ul
1-1=916.4
1-2=423.0
2-1=934.9
2-2=737.0
Day-1=322.9
Day-2=412.4
Day-3=403.1
Day-4=729.1
Night-1=152.2
Night-2=314.2
Night-3=276.7
Night-4=202.4
