LECTURE 8 - RNA ISOLATION

what are some reasons we isolate RNA

cDNA library source

Northern blotting

reverse transcriptase PCR

nuclease protection assays

what must we treat all solutions for

we treat all solutions to remove RNases

what do we treat non-amine solutions with

diethyl-pyrocarbonate

what is RNasin

RNase protein inhibitor

what are other ways we can control RNase activity

treat glassware

disposable gloves

wash benches with RNase zap and ethanol

why does it matter where our tissue sample is from

because some tissues like the spleen and liver have a higher RNase content

what do we use to preserve the integrity of our RNA

liquid nitrogen

or we keep it at lower temperatures

how long can we store RNA at different temperatures

a day or less at 37*C

1 month at 4*C

as long as needed at -20*C

explain the steps in manual RNA extraction

1. suspend cells in homogenisation mixture

2. homogenise tissue so cells separate

3. add RNA extraction buffer (37*C for 30min)

4. extract using phenol/chloroform

5. precipitate RNA

6. resuspend RNA using RNase free DNase 1, RNase inhibitor, MgCl2 and DTT (37*C for 60min)

7. do a phenol/chloroform extraction again

8. resuspend in tris EDTA

what does the homogenisation mixture contain

NaCl

MgCl2

Tris

Nonidet - p40

DTT

Rnase inhibitor

what does an RNA extraction buffer contain

Tris (pH 8)

EDTA

NaCl

SDS

Proteinase K

what are other methods of RNA isolation

using guanidine thiocyanate

trizol

how does guanidine thiocyanate work

it effectively lyses cells and inactivates RNases at the same time

how does Trizol work

it isolates DNA, RNA and proteins

explain how an RNA extraction column works

we first disrupt cells using guanidine isothiocyanate

the lysate is then diluted with ethanol and applied to the RNA binding matrix

proteins and contaminants will be removed via washes

then we elute the total RNA

how much of total RNA is mRNA

about 3%

what feature of mRNA allows us to separate it from total RNA

the polyA tail

does all mRNA have a polyA tail

no, bacterial mRNA does not have a polyA tail

explain how the polyA tail allows us to separate out mRNA

we use a column that contains a synthetic oligonucleotide; dTTP

the polyA tail of mRNA hybridises to the dTTP

we can wash away any unbound RNA

we then use a hot buffer to elute the mRNA

we then purify again

why do we use a hot buffer to elute mRNA

the hot buffer provides thermal energy to break the hydrogen bonds between the dTTP and the polyA tail

why do we purify mRNA twice

because some rRNA binds to dTTP by chance, so we want to rid of this