RNA EXTRACTION

involves the isolation of RNA molecules from a biological

sample while removing contaminants such as proteins, DNA, and other cellular debris.

rna extraction

STEPS in RNA EXTRACTION

1. Sample collection

2. cell lysis

3. Phase Separation (Organic Extraction Method)

4. RNA Precipitation

5. RNA resuspension

commonly used in Sample Collection and Preservation

rnalater or freezing in liquid nitrogen

to disrupt cellular membranes and inactivate RNases.

detergents or chaotropic agents

detergents or chaotropic agents examples

guanidium, isothiocyanate

help release RNA from the cells.

Mechanical disruption

Mechanical disruption methods

sonication, bead beating, or homogenization,

Phase Separation most common method involves

phenol-chloroform extraction, using reagents like TRIzol.

Phenol-chloroform creates a biphasic separation (AIO)

Aqueous phase (RNA)

Interphase (proteins)

Organic phase (lipids and DNA)

RNA is precipitated by adding ______ in the presence of salts like sodium acteate

isopropanol or ethanol

is obtained by centrifugation and washed with 70% ethanol to remove

rna pellet

The RNA pellet is air-dried and resuspended in

rnase free water and te buffer

Three commonly used methods can effectively isolate RNA from samples:

1. the guanidinium thiocyanate-based method

2. Spin column-based method

3. the combined guanidinium thiocyanate-column based method

the cell extract is extracted with phenol/chloroform at low pH

Guanidinium Thiocyanate Phenol-Chloroform Extraction

chaotropic agent used in protein degradation.

guanidium thiocyanate

guanidium thiocyanate how does it work

disrupts the cells, denatures the proteins, and deactivates the

nucleases,

is separated from DNA after extraction with acidic solution

consisting guanidinium thiocyanate and phenol

rna

Principle of the Spin Column-Method

Selective RNA Binding to Silica Membrane Impurity Removal via Wash Steps RNA Elution

This method combines guanidinium thiocyanate-based RNA

denaturation with silica membrane column purification for

efficient and high-purity RNA isolation.

3. Guanidinium Thiocyanate-Column Based RNA Extraction

effectively lyses cells and inactivates RNases, preventing RNA degradation.

guanidine thiocyanate

selectively binds RNA to silica membranes, removing contaminants like DNA, proteins, and lipids.

spin column based extracion

It is the template for protein translation, and most of the eukaryotic

mRNAs carry tracts of it at their 30 termini.

isolation of poly a rna

Two methods are commonly used in the selection of poly(A)+ isolation

column chromatography on oligo(dT) columns and batch chromatography

is normally used for the purification of large quantities (>25μg) of poly(A)+ RNA isolated from mammalian cells.

Column Chromatography

is the preferred method when working with small amounts (<50μg) of total mammalian RNA.

Batch Chromatography

Batch Chromatography carried out wiht a

fine grade oligo(dt) cellulose

an alternative for the purification of poly(A)+ RNA from total RNA sample.

magnetic oligo(dt) bead

Degrades single-stranded RNA but notDNA

rnase a

Specifically degrades RNA in RNA-DNA hybrids

rnase h

Cleave RNA at specific nucleotide sequences

rnase I

Spin column kits example

qiagen, zymo

selectively precipitates RNA while

leaving DNA in solution

lithium chloride