RNA ISOLATION

Rna isolation

Fundamental laboratory technique that involves extracting ribonucleic acid from biologic sample by separating it from proteins, dna, lipids and other cellular debris for molecular testing

Unstable

Rna is highly what molecule?

Strict RNAse free condition

Rna isolation requires what kind of condition?

Strong denaturants

What can be used to inhibit endogenous RNAses?

GITC (guanidinium isothiocyanate)

What is the most common reagent?

Rnase

Enzyme that break down RNA into smaller components by cutting the bonds between nucleotides

1. Sample collection

2. Cell lysis

3. Removal of contaminants

4. RNA purification

5. Elution

Rna isolation steps

Chloroform

In removal of contaminants, what is added to the lysate

Upper aqueous phase

Lower organic phase

Under acidic conditions, centrifuged lysate is separated into two distinct phases

Upper aqueous phase

What phase contains the target RNA?

Lower organic phase

What phase contains proteins, genomic DNA and cellular lipids?

rRNA

Most abundant RNA in the cell

mRNA

Carries genetic information for protein synthesis

tRNA

Delivers amino acids during translation

Total RNA

Present in all RNA extracted from a cell

Organic reagents

Main principle of RNA organic method

Silica column/magnetic beads

Main principle of RNA solid phase method

Trizol/phenol and guan

Common reagent of organic RNA isolation

Silica membrane and magnetic bead

Common reagent of Solid-phase RNA isolation

Organic RNA isolation

Uses TRIZOL reagent

Organic RNA isolation

Requires hazardous chemicals

Solid-phase RNA isolation

Uses silica columns;

Easily automated;

Commonly used in clinical molecular laboratories;

Uses magnetic beads

Solid phase rna isolation method

A hospital laboratory is performing RT-PCR to detct SARS-CoV-2. The laboratory must process hundrends of patient specimens daily using an automated extraction system.

Which RNA isolation method is most appropriate?