Week 8 RNeasy RNA Extraction Reagents

In the name, duh

What are the reagents used in the RNeasy MiniPrep?

RNAprotect Bacteria Reagent, TE lys proK (aka Lysis Buffer), Buffer RLT (aka lysis buffer RLT), Wash Buffers RW1 and RPE, Elution Buffer

What is the purpose of using RNAprotect Bacterial Reagent?

• It immediately enables RNase inactivation

• Stabilizes RNA

• Preserves RNA expression pattern

• Prevents gene induction/down-regulation

Why is the use of RNAprotect Bacteria Reagent important?

Since enzymes degrading RNA leads to losing many transcripts, since bacterial mRNA has a very short half life

What are the components of the lysis buffer (TE lys proK)?

Tris buffer (contains EDTA), Lysozyme, Detergent, and Proteinase KWha

What is the purpose of Proteinase K in the TE lys proK lysis buffer?

• Help release nucleic acids

• Deactivate nucleases

• Prevent nucleic acids from nuclease attack

What is the function of nucleases?

NOT A REAGENT

They degrade nucleic acids (not wanted for this experiment, this is why we use Proteinase K)

Is the RNA of a harvested biological sample very stable or unstable?

Extremely unstable

What are the components of lysis buffer (RLT)?

Beta-Mercaptoethanol and Chaotropic Salt (Guanidium Isothiocyanate)

What is the chaotropic salt used in this experiment?

Guanidium Isothiocyanate

What is the purpose of beta-mercaptoethanol in the lysis buffer (RLT)?

To degrade ribonucleases by breaking disulfide bonds (makes proteins unfold and become non functional)

What happens to a protein if it’s disulfide bonds are broken?

The protein unfolds and becomes non-functional

How are ribonucleases degraded in this experiment?

Using beta-mercaptoethanol, which breaks the disulfide bonds

What is the purpose of using guanidium isothiocyanate?

To maintain a hydrophobic solution by preventing H bonds between RNA and water

To set up conditions for the RNA to bind to the membrane

What are the two wash buffers used in this experiment?

Buffers RW1 and RPE

What do the wash buffers contain?

Wash buffer and 90% EtOH

What is the purpose of ethanol in the wash buffers?

• Salts and small organic molecules dissolve in the ethanol and are centrifuged out

What does the elution buffer contain?

RNAse free water

What is the purpose of using RNAse free water?

To rehydrate RNA and free it from the silica membrane for elution