Dr. Adrian “Fritz” Gombart, Office: 2135 ALS Office hours ...

RNA Isolation and Northern Blot Analysis

Dr. Adrian "Fritz" Gombart, Office: 2135 ALS Office hours: Wednesdays 1-3pm Phone: 737-8018 adrian.gombart@oregonstate.edu

Experiment 3: Isolation of total RNA from eukaryotic tissue culture cells

for analysis of gene expression Why?

Central Dogma of Molecular Biology

Flow chart of Experiment 3

Teams of 2 Prepare RNA samples ? Feb. 2/3 Teams of 4 run formaldehye-agarose gel ? Feb. 7/8

Northern transfer ? Feb. 7/8 Probe preparation ? Feb. 9/10 Prehybridization ? Feb. 14/15

Hybridization ? Feb. 14/15 Post-hybridization washing ? Feb. 16/17

Signal detection ? Feb. 16/17

Different methods to isolate RNA

Guanidine isothiocyanate lysis and centrifugation through CsCl gradient

- high quality RNA >200 nucleotides; lengthy protocol

Guanidine isothiocyanate lysis and centrifugation through silica resin column

- high quality RNA >200 nucleotides; quick protocol

Trizol reagent (phenol/chloroform based); precipitation from aqueous phase

- high quality RNA >200 and 200 nucleotides; lengthy protocol

Guanidine isothiocyanate lysis and centrifugation through silica resin column

- high quality RNA >200 nucleotides; quick protocol

Trizol reagent (phenol/chloroform based); precipitation from aqueous phase

- high quality RNA >200 and ................
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