RNA EXTRACTION RNA extraction is the purification of RNA from biological samples.

DNA --------------> mRNA --------------> protein

Lots of information in mRNA:

When is gene expressed?What is timing of gene expression?What is the level of gene expression?

(but what does an mRNA measurement really say about expression of the protein?)

10-5 micrograms RNA

80-85% is ribosomal RNA15-20% is small RNA (tRNA, small nuclear RNAs)

About 1-5% is mRNA

-- variable in size-- but usually containing 3’ polyadenylation

RNA in a typical eukaryotic cell:

RNA is chemically unstable -- spontaneous cleavage of phosphodiester backbone via intramolecular transesterification

RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases)

RNases are released upon cell lysisRNases are present on the skinRNases are very difficult to inactivate

-- disulfide bridges conferring stability-- no requirement for divalent cations

for activity

The problem(s) with RNA:

Common sources of RNase and how to avoid them

Contaminated solutions/buffers

USE GOOD STERILE TECHNIQUETREAT SOLUTIONS WITH DEPC (when possible)MAKE SMALL BATCHES OF SOLUTIONS

Contaminated equipment

USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGSBAKE GLASSWARE, 300°C, 4 hoursUSE “RNase-free” PIPET TIPSTREAT EQUIPMENT WITH DEPC

INHIBITORS OF RNASE DEPC: diethylpyrocarbonate

Alkylating agent, modifying proteins and nucleic acids

Fill glassware with 0.1% DEPC, let stand overnight at room temp. or NaOH or H2O2

Solutions may be treated with DEPC (0.1%), then autoclave (DEPC breaks down to CO2 and ethanol)

Vanadyl ribonucleoside complexes Competitive inhibitors of RNAses, but need to be removed from

the final preparation of RNA

Protein inhibitors of RNAse horseshoe-shaped, leucine rich protein, found in cytoplasm of most mammalian tissues must be replenished following phenol extraction steps

Making and using mRNA (1)

Making and using mRNA (2)

Separate WBCs from RBCs, if necessary

Lyse WBCs or other nucleated cells in presence of protein denaturants, RNase

inhibitors

Denature/digest proteins

Separate proteins, DNA, and contaminants

from RNA

Precipitate RNA if necessary

Resuspend RNA in final buffer

BASIC STEPS IN ISOLATINGRNA FROM CLINICAL SPECIMENS

SELECTIVE CAPTURE OF MRNA Oligo dT is linked to cellulose matrix

RNA is washed through matrix at high salt concentration Non-polyadenylated RNAs are washed through polyA RNA is removed under low-salt conditions (not all of the non-polyadenylated RNA gets removed

Poly(U) sepharose chromatography Poly(U)-coated paper filters

Streptavidin beads: A biotinylated oligo dT is added to guanidinium-

treated cells, and it anneals to the polyA tail of mRNAs Biotin/streptavidin interactions permit isolation of the

mRNA/oligo dT complexes

RNA ISOLATION METHODSCESIUM CHLORIDE GRADIENT Used mainly to get clean RNA for Northern

blots Homogenize cells in guanidinium

isothiocyanate and b-mercaptoethanol solution.

Add to CsCl gradient and centrifuge for 12–20 hours; RNA will be at the bottom of tube.

Re-dissolve in TE/SDS buffer. Precipitate RNA with salt and ethanol, then

rehydrate. Advantage: high quality Disadvantages: extremely time-consuming,

hazardous materials disposal issues

RNA ISOLATION METHODSGUANIDINIUM-BASED ORGANIC ISOLATIONGUANIDINIUM THIOCYANATE-PHENOL-CHLOROFORM EXTRACTION IS THE MOST COMMON METHOD. (TRIZOL METHOD).

Phenol/guanidinium (chaotropic ) solution disrupts cells, solubilizes cell components, but maintains integrity of RNA.

Add chloroform, mix, and centrifuge. Proteins/DNA remain at interface. RNA is removed with aqueous top layer. RNA is precipitated with alcohol and rehydrated. Advantage:

faster than CsCl method better purity no RNA is lost

Disadvantages: fume hood required, hazardous (Phenol and chloroform) waste disposal issues, chaotropic

liquid-liquid extraction technique

RNA ISOLATION METHODSNONORGANIC SALT PRECIPITATION

Cell membranes are lysed and proteins are denatured by detergent (such as SDS) in the presence of EDTA or other RNase inhibitors.

Proteins/DNA are precipitated with a high concentration salt solution.

RNA is precipitated with alcohol and rehydrated.

Advantages: Fast and easy, nontoxicProduces high quality RNA

RNA ISOLATION METHODS AFFINITY PURIFICATION OF RNA Lyse cells, and spin to remove large

particulates/cell debris Apply lysate (containing nucleic

acids and cellular contaminants) to column with glass membrane

Wash with alcohol to remove contaminants; nucleic acids stick to glass membrane while contaminants wash through. Treat with DNase enzyme to remove contaminating DNA.

Apply water to the column; purified RNA washes off the glass and is collected

Disadvantage cannot adsorb RNA transcripts shorter

than 200 bp (siRNA and miRNA)

NUCLEIC ACID ANALYSIS

DNA or RNA is characterized using several different methods for assessing quantity, quality, and molecular size. UV spectrophotometry Agarose gel electrophoresis Fluorometry Colorimetric blotting

QUANTITY FROM UV SPECTROPHOTOMETRY DNA and RNA absorb maximally at 260 nm. Proteins absorb at 280 nm. Background scatter absorbs at 320 nm.

QUANTITY FROM UV SPECTROPHOTOMETRY [DNA] =(A260 – A320) X dilution factor X 50 µg/mL [RNA] =(A260 – A320) X dilution factor X 40 µg/mL Concentration = µg of DNA or RNA per mL of

hydrating solution

Multiply the concentration of theDNA or RNA sample by the

volume of hydrating solution added.

Example for DNA: 150 µg/mL X 0.1 mL = 15 µg

Concentration from UV Spec. (µg DNA

per ml of hydrating solution)

Volume of hydration solution

DNA yield

QUANTITY FROM UV SPECTROPHOTOMETRY CALCULATING YIELD

A260/A280 = measure of purity

(A260 – A320)/(A280 – A320)

1.7 – 2.0 = good DNA or RNA

<1.7 = too much protein orother contaminant (?)

QUALITY FROM UV SPECTROPHOTOMETRY

QUALITY FROM AGAROSE GEL ELECTROPHORESIS Genomic DNA:

0.6% to 1% gel, 0.125 µg/mL ethidium bromide in gel and/or in running buffer

Electrophorese at 70–80 volts, 45–90 minutes. Total RNA:

1% to 2% gel, 0.125 µg/ml ethidium bromide in gel and/or in running buffer

Electrophorese at 80–100 volts, 20–40 minutes.

RNA SIZE AND QUALITY FROMAGAROSE GEL ELECTROPHORESIS Size: mRNA may be smaller or larger than

ribosomal RNA (rRNA). Quality: High-quality RNA has these

characteristics: 28S rRNA band : 18S rRNA band = 2:1 intensity Little to no genomic DNA (high MW band)

Note: If 18S rRNA is more intense than 28S rRNA, or if both bands are smeared, RNA degradation is probable.

Use radiolabelled poly dT in a pilot Northern hybridization--should get a smear from 0.6 to 5 kb on the blot

Degraded RNA

DNA

28S18S

5S rRNA, tRNA, and other small RNA molecules

mRNA = background smearhigh low MW

100 50 25 ngGenomic DNA

markers

CULTURED CELL RNA

STORAGE CONDITIONS

Store DNA in TE buffer at 4 °C for weeks or at –20 °C to –80 °C for long term.

Store RNA in RNase-free ultra pure water at –70 °C.

TROUBLESHOOTING NUCLEIC ACIDPREPARATION METHODS Purifying RNA: the key is speed Problem: No or low nucleic acid yield.

Make sure that ample time was allowed for resuspension or rehydration of sample.

Repeat isolation from any remaining original sample (adjust procedure for possible low cell number or poorly handled starting material).

Concentrate dilute nucleic acid using ethanol precipitation.

TROUBLESHOOTING NUCLEIC ACIDPREPARATION METHODS Problem: Poor nucleic acid quality

If sample is degraded, repeat isolation from remaining original sample, if possible.

If sample is contaminated with proteins or other substances, clean it up by re-isolating (improvement depends on the extraction procedure used).