Quantitative real-time RT-PCR sample preparation: Day 1 of 2

Notes:

The MJ Research PTC-200 Thermal Cycler was used for all reactions. A heated lid was used to minimize evaporation on long incubations. This machine can easily obtain temperatures from 4-100 °C and has a very rapid ramp time. The procedures that require removal to ice were simply allowed to drop to 4°C.

The following total RNA isolation protocol is a slight modification of that suggested by the manufacturer of TRIzol (Invitrogen).

Sample Preparation

Partition

• In a 2 ml screw cap tube with o-ring:
• Add 250 uL Resuspended Cells in PBS
• Add 750 uL TRIzol LS (1 ml/ 10⁷ cells)
• Add 800 uL Chloroform
• Shake vigorously for 15 seconds
• Let stand for 10 min
• Centrifuge at 4 °C for 15 minutes, 12,000 x g
• Aliquot the top aqueous layer into a new 1.5 ml tube. (The DNA is in between the two phases; do not take this white precipitate.)

Precipitation

• Add 1 uL glycogen or NF pellet paint (Novagen)
• Add equal volume 2-Propanol
• Invert to mix the layers
• Let stand for 10 min
• Centrifuge at 4 °C for 15 minutes, 12,000 x g
• Carefully remove supernatant and then quick spin
• Remove as much 2-Propanol as possible with P10 Tip

Wash pellet

• Add 1 mL 70-75% ethanol
• Centrifuge at 4 °C for 15 minutes, maximum speed
• Carefully remove supernatant and then quick spin
• Remove as much ethanol as possible with P10 Tip
• Air dry in the hood for 10 minutes

Resuspend

• If there was more than 1 tube per condition, divide the volume of water between them to resuspend, then combine.
• Add 87 uL DEPC water
• Quick vortex to mix
• Heat 60 °C for 10 minutes
• Can be stored at -80 °C

DNase treatment

• Aliquot the sample into a 0.5 ml tube
• Add 87 uL sample (in water)
• Add 10 uL DNase Buffer:
• 50 mM Tris-HCL
• 5 mM MgCl₂ (pH 7.5)
• Add 3 uL DNase I / RNase free
• Thermal cycler conditions:
• 37 °C, 30 min
• 75 °C, 5 minutes
• 4 °C, hold

Precipitation

• Add 100 uL DEPC water to sample and mix
• Transfer sample to 1.5 mL tube
• Add 1 uL glycogen or NF pellet paint (Novagen)
• Add 20 uL 3M Sodium Acetate
• Add 400 uL ethanol
• Invert to mix
• Store at -20 °C overnight (1 hour minimum)

Quantitative RT-PCR (TaqMan) Analysis: Day 2 of 2

Wash pellet

• Centrifuge at 4 °C for 20 minutes, maximum speed
• Carefully remove supernatant
• Add 750 uL 70% ethanol
• Centrifuge at 4 °C for 15 minutes, maximum speed
• Carefully remove supernatant

Repeat Wash

• Add 750 uL 75% ethanol
• Centrifuge at 4 °C for 15 minutes, maximum speed
• Carefully remove supernatant, then quick spin
• Remove as much ethanol as possible with P10 Tip and then air dry in the hood for 10 minutes

Resuspend

• Add 20-50 uL water
• Heat at 60 °C for 10 minutes
• an be stored at -80 °C

Quantitate the RNA yield

• Dilute samples for Optical Density reading in a 0.5 ml tube
• Add 1 uL sample
• Add 79 uL 1X TE
• Measure and calculate the following:
• A260
• A280
• Concentration
• Purity
• Dilution factor ( (sample + water)/sample )
• Stock concentration (concentration * dilution factor)
• Total volume (total volume * stock concentration)
• Yield

Gel Analysis of RNA (optional)

• Pour a 1% agarose gel; use 10 tooth comb:
• Add 1.25 g agarose
• Add 1.25 mL 1X TBE + ethidium bromide
• Run 1 kB ladder:
• Add 1 uL Gibco-BRL 1 KB ladder
• Add 30 uL Dilution and Loading Buffer
• Make sample dilutions:
• Add 20 uL DF 40 diluted sample used for OD
• Add 5 uL Bottom Only 6X loading dye
• Load wells and run gel at constant voltage: 100 V
• Visualize under UV transillumination

First strand (For a 20 uL reaction)

• To 0.5 ml tube, add 1-10 uL 10 ug of RNA
• Add 1 uL T7-Poly dT primer or random hexamer primer (250 ng/uL)
• Add QS with water to 14 uL
• Mix, spin, then incubate
• Thermal cycler conditions:
• 70 °C, 10 minutes
• 4 °C, 2 minutes
• Quick spin
• Add 2 uL 10X Stratascript buffer (stratagene)
• Add 2 uL 0.1 M DTT
• Add 1 uL 10 mM dNTP mix
• Mix, spin, then incubate
• Thermal cycler conditions:
• 42 °C, 2 minutes
• Add 1 uL Stratascript RT enzyme (Stratagene) (total volume 20 uL)
• Mix, spin, then incubate
• Thermal cycler conditions:
• 42 °C, 60 minutes
• 95 °C, 5 minutes
• 4 °C, hold
• Dilute cDNA to 100 ng/uL final concentration for Real-time PCR analysis