MBTE4034

METHODS IN MOLECULAR
BIOTECHNOLOGY LAB II
RNA extraction
RNA extraction
• Controlling RNAse activity
• RNAse inactivation
• DEPC
• Inactivates RNAse enzymes;
• Reagents prepared by incubating with 0.1%
diethylpyrocarbonate(DEPC) and then autoclaved to get rid of active
DEPC
• Remove protein
• Phenol/Chloroform
• Isolate RNA from DNA
• Removal of DNA
Isolation of RNA with Guanidinium Buffers
• Lysis buffer contains
guanidium thiocyanate
(chaotropic salt)
• Denature proteins (RNAse
inc.)
Phenol/Chloroform extraction
• Lysis buffer
• Water-saturated phenol
• Cell lysis
• Chaotropic salts (e.g. guanidium thiocyanate)
• Denature proteins
• Acidic phenol/Chloroform
• Denaturing proteins and dissolves lipids
• Acidic to aid partitioning of RNA into aqueous
phase during separation.
Phenol/Chloroform extraction (cont)
• High pH cause RNA and DNA isolated in the same fraction
• Low pH isolates RNA from DNA and proteins
Qiagen RNeasy Mini Kit
Qiagen RNeasy Mini Kit (cont)
• RLT buffer
• High concentration of guanidine isothiocyanate, supports RNA
binding to silica membrane in spin column
• RW1 buffer
• Guanidine salt with ethanol
• Stringent wash buffer removes biomolecules non-specifically
bound to membrane, remains RNA > 200bp
• RPE buffer
• Mild washing buffer removing traces of salt remains
RNA quality
Caution dealing with RNA
RNAse are extremely stable and requires no cofactor to function
1. Clean pipettes with
RNaseZap and Kimwipe 3. Always wear
gloves, clean with
RNaseZap

4. Clean bench surface with RNaseZap

2. Use unopened tips for RNA extraction and

cDNA synthesis. No reusing tips.