RNA EXTRACTION

GROUP 2 3MT-3 ARNAN BADONG BALUYOT BASTIDA BAUTISTA BERMUDEZ

 RNA (Ribonucleic acid) is a

polymeric substance present in living cells and many viruses, consisting of a long singlestranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is used in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses

Objective
Understand the development of RNA

extraction. Understand why we isolate RNA, the most critical step in performing most of the molecular biology experiments. Know ways of inactivating RNAses Understand the basic principles in isolating total RNA and mRNA

Historical Background
First used of guanidinium chloride in isolation

of RNA described by by Volkin and Carter(1951) Grassmann and Defner described phenol at extracting proteins(1953) Kirby demonstrated the use of phenol to seperate nucleic acids from proteins in 1956.

Historical Background
The interest for using guanidinium chloride

was renewed by Cox and friends during the 1960s. Guanidinium extractions became the method of choice. In 1977, Ullrich et al. mentioned the used of guanidinium thiocyanate.

Historical Background
Chirgwin et al. used guanidinium thiocyanate

to isolate undegraded RNA from ribonuclease-rich tissues like pancreas.

A combination of guanidinium thiocyanate

and hot phenol for RNA isolation was reported by Feramisco et al. in 1981

Historical Background
In 1987, Chomczynski and Sacchi combined

guanidinium thiocyanate with phenolchloroform extraction under acidic conditions

Chomczynski & Sacchi method has been the

method of choice to isolate RNA from cultured cells and most animal tissues.

Principle
guanidinium isothiocyanate (powerful

protein denaturant) -> inactivation of RNases acidic phenol/chloroform -> partitioning of RNA into aqueous supernatant for separation
Note: low pH is crucial since at neutral pH

DNA and not RNA partitions into the aqueous phase. Check the pH of old TRIZOL/TRI reagents!

Reagents
TRIzol or TRI reagent 0.8 M sodium citrate / 1.2 M NaCl isopropanol (2-propanol) chloroform 75% EtOH in DEPC H2O RNase free water (filtered or DEPC)

Procedure
1. Homogenization 2. Phase Separation 3. RNA Precipitation 4. RNA Wash 5. Redissolving RNA

1. Homogenization
Time Required: Cell lysis only takes a few

minutes per well, but tissue homogenisation can take 10-20 minutes per sample depending on how tough the tissue is.
(PBS wash) add trizol (cell lysis) 1ml / 3.5 cm diameter well (6-well)5ml / 75

ml bottlehomogenise by pipetting several times (mechanic lysis)

1. Homogenization
homogenise by pipetting several times

(mechanic lysis)
alternative for tubes: vortex 1 min alternative for tissue: grind 1 g tissue in liquid nitrogen in a mortar and pestle, put tissue into plastic screw-cap centrifuge tube + 15 ml TRIzol reagent, incubate samples for 5 min at room temp or 60 C (scaled up as needed)

(5min at RT for complete dissociation of

nucleoprotein complexes)

2. Phase Separation
Time Required: 15-45 min depending on number of

samples and whether an additional chloroform wash is necessary. add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml) shake for 15 sec (do not vortex) incubate 2-5 min at RT spin max. 12000g, 5-15 min, 2-8C if centrifugation hasn't been sufficient the DNAcontaining interphase will be cloud-like and poorly compacted

2. Phase Separation
Optional Step: If supernatant appears turbid an additional chloroform cleaning step can be inserted here. transfer aqueous upper phase into new tube Difficult Step: Take care not to aspirate the DNA-containing white interface. This quickly happens and will lead to DNA contamination in your RNA prep.

TRIZOL phases after chloroform addition

TOP - colourless aqueous phase (RNA) - 60% TRIZOL volume MIDDLE - interphase (DNA) BOTTOM - red (organic) phenol-chloroform phase (proteins & lipids)

3. RNA Precipitation
Time Required: 20-40 min depending on number of samples
add isopropanol (70% of aqueous phase or 1/2 trizol volume)

0.8 M sodium citrate or 1.2 M NaCl can be added

(incubate 10min at RT) spin max g, 10-15 min, 4C remove supernatant

4. RNA Wash
Time Required: 15-30 min depending on number of samples
wash pellet 70% EtOH (add & vortex briefly) 70% ethanol prepared with RNase-free water

Optional Step some prefer to wash the pellot more than once with 70% ethanol

4. RNA Wash
spin max g, 2-10 min, 4C air-dry pellet for 5-10 min Critical Step Do not

overdry the pellet or you won't be able to redissolve it.

4. RNA Wash
Optional Step add RNase inhibitor Optional Step incubate at 55-60 C for 10 min if hard to redissolve
transfer to eppendorf tube spin 4 C, 5 min (to pellet undissolved

material)

5. Redissolving RNA
dissolve pellet in 50-100 l filtered or DEPC

H2O (note: DEPC inhibits RT reaction) alternatively, 0.5% SDS pipetting up and down, heat to 55-60C for 10 min

Common mistakes
use too little trizol; very small volumes are

hard to separate and will most likely lead to contamination aspirate some white interphase (DNA) when removing aqueous supernatant (RNA) use phenol/chloroform of the wrong pH (has to be acidic) not working under the hood (phenol is toxic, chloroform is a narcotic)

The following guidelines should be observed carefully while working with RNA:
Always wear disposable gloves. Usually our skin contains many bacteria and molds that can contaminate the RNA preparation and be a source of RNases. Good and clean microbiological techniques will

prevent microbial contamination. Use sterile, disposable plastic ware and automatic pipettes reserved for RNA work to prevent crosscontamination with RNases from shared equipment.

Application
CLINICAL AGRICULTURE FORENSIC RESEARCH APPLIED

Sources
http://amrita.vlab.co.in/index.php?sub=3&brch=186

&sim=718&cnt=1 http://www.invitrogen.com/site/us/en/home/brands/ ambion.html http://openwetware.org/wiki/RNA_extraction_using _trizol/tri http://www.labturbo.com/index.php?op=application http://physiology.med.cornell.edu/faculty/mason/lab /zumbo/files/PHENOL-CHLOROFORM.pdf http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789 530/

Member Contributions
Arnan- Applications Badong-Methodology Baluyot-Historical Background Bastida-Applications Bautista-Video Presentation and Reporter Bermudez-Video Presentation and Reporter