6.

0 RNA isolation and Reverse Transcription PCR Date:

6.1 RNA isolation of adherent cells by TRIzol reagent Notes
Reagents: Samples: Do RNA isolation as first experiment
TRIzol
Chloroform (RNA zone)
Isopropyl alcohol (ispropanol)
75% Ethanol
Glycogen (20 mg/ml)
Nuclease free water
Apparatus:
Centrifuge
PCR machine (509)

A. Preparing samples

Homogenizing samples (Adherent cells – Monolayer)

1. Remove growth media from culture dish
2. Add TRIzol reagent
TRIzol Dish
1 ml 35 mm
3 ml 60 mm
8 ml 100 mm
3. Lyse cells directly in culture dish by pipetting cells up & down

Homogenizing samples (Cell suspension)

1. Remove growth media after centrifuge, leaving the cell pellet
2. Add TRIzol reagent
TRIzol Cell suspension
750 ul 250 ul 5-10 x 10^6 cells
Total 1 ml
3. Lyse cells pipetting up & down several times (properly homogenize!)

Proceed to phase separation (improve RNA yield), or store homogenized sample.

Store at RT for several hours @
Store at -60 to -70 oC (1 month)

Phase separation
1. INCUBATE at RT = 5 min
2. Add 200 ul chloroform. Shake tube vigorously to mix. INCUBATE at RT = 2-3 min
SPIN 13.5k rpm (10-15 min, 4oC)
3. Transfer ~500 ul aqueous layer (upper) into new tube [angle tube to 45o]
4. Add 500 ul chloroform. Shake tube vigorously to mix. INCUBATE at RT = 2-3 min
SPIN 13.5k rpm (10-15 min, 4oC)
#STEP to remove phenol & DNA completely
5. Transfer aqueous layer (upper) into new tube
B. RNA isolation procedure

RNA precipitation
1. Add 500 ul isopropanol, vortex to mix
2. Add 2 ul Glycogen (final: 2mg/ml), vortex to mix (cloudy)
3. INCUBATE for 10 minutes
4. SPIN, 13.5k rpm (30 min, 4oC), discard supernatant (just pour)
Leave only RNA pellet (whitish)
RNA wash
1. Wash with 1 ml 75% EtOH (per 1 ml of TRIzol)
2. SPIN, 13.5k rpm (5 min, 4oC), remove supernatant (EtOH)
3. Microspin, remove excess supernatant (EtOH) using 100 ul pipette
4. Air-dry: RNA (whitish)  RNA (clear, but still can see) (5 – 10 min)

RNA suspension
1. Add 10 ul Nuclease-free water, resuspend pipet up & down (based on pellet amount)
2. Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.
3. Check concentration (Nanodrop)
4. Typical RNA (A260/280 of >1.8) yields from various starting materials

6.2 DNAse I Treatment of Total RNA (ReverTra Ace, Toyobo)

Place mixture in PCR machine
Prepare: 25 mM EDTA Parameters:
Set-up PCR machine Incubate: 37oC, 20 min
PAUSE: Add 1 ul 25 mM EDTA
Mix following reagents: (Prepare master mix buffer, DNaseI, MQ) (Total vol: 11 ul)
Incubate: 70oC, 10 min
Nuclease-free water X ul
Total RNA (1 ug) Y ul Proceed with Reverse transcription
10x DNase I buffer 1 ul PCR (RT-PCR)
RNase-free DNase I (10 U/ul) 0.5 ul
10 ul

6.3 Reverse transcription PCR (RT-PCR) (ReverTra Ace, Toyobo)

1. Annealing primers PCR Parameters for incubation:

30oC, 10 min
Total RNA (after DNaseI treatment) 11 ul 42oC, 50 min
Oligo (dT) [50 mM] 2 ul 5 mM
99oC, 5 min
13 ul

Incubate, 65oC, 5 min Store reacted solution at 4 oC (short

Finish: Quickly put ON ICE, > 1 min term) or -20 oC (long term)

2. Add these reagents to Total DNA + Oligo mixture

Total DNA + Oligo mixture 13 ul

5x RT buffer 4 ul 1x
dNTP [10 mM] 2 ul 1 mM
ReverTra Ace 1 ul
20 ul