RNA extraction

A single-phase guanidinium thiocyanate and acid phenol reagent lyses the sample and denatures its RNases in one step; chloroform then splits the mixture so that DNA and protein leave with the organic phase and the interphase while RNA stays aqueous, and isopropanol brings the RNA down.

ng/µLSimulator readyCore simulatorEngine: Liquid handling & reaction assembly

Choose a sub-technique

In learning pathsMeasure gene expression · step 1 of 5 · next: RNA integrity check →

Our ruleSimulations prepare you for the bench but don't replace hands-on practice.

Sub-technique you chose

TRIzol

How it differs from the standard method

Keeps the whole size range, from 28S down to miRNA, if the precipitation is given time in the cold, which is its main advantage over a standard column.
No binding capacity to saturate, so it scales from a needle biopsy to a gram of tissue; what limits it is the tissue per millilitre of reagent, about 30 mg, because the sample itself dilutes the chaotrope.
The interphase is the hazard: pipetting into it puts phenol and genomic DNA straight back into the sample, and phenol stops reverse transcriptase outright.
Phenol is corrosive and the reagent contains guanidinium thiocyanate, so it needs a fume hood and must never meet bleach.

In this lesson · about 7 min read

The technique is mostly about keeping RNase out
Guanidinium lyses; acid phenol is what makes it RNA
What the readout actually is
Columns have a capacity, and a size cut-off you did not ask for
Residual genomic DNA, and why it matters for anything that amplifies
Key terms
When to use it
Sub-techniques

1The technique is mostly about keeping RNase out

A DNA prep is a purification problem. An RNA prep is a purification problem with a clock running, because the enzymes that destroy the product are already in the room.

2Guanidinium lyses; acid phenol is what makes it RNA

Guanidinium thiocyanate is the most powerful of the common chaotropic salts. At molar concentrations it disrupts the ordered water around proteins, so membranes dissolve, nucleoprotein falls apart and every nuclease in the sample is denatured in the same motion that releases the RNA.

Lyse

Guanidinium thiocyanate

Dissolves membranes, strips protein off nucleic acid and denatures the RNases in the same step. The sample is protected from the moment it is in the reagent, and not before.

Partition

Acid phenol and chloroform

At pH 4.5 DNA goes to the organic phase and the interphase while RNA stays aqueous. At neutral pH both stay aqueous, and the method stops being RNA-specific.

Recover

Alcohol or silica

Isopropanol precipitation keeps the whole size range, including miRNA. A silica membrane is faster and cleaner but keeps only what the ethanol content tells it to.

3What the readout actually is

An RNA prep is reported as ng/µL, but that number alone is almost useless, because three independent things can be wrong with a tube of RNA and concentration only sees one of them. Absorbance at 260 nm counts RNA, genomic DNA, free nucleotides and anything else that happens to absorb there, so it reads high on a prep that carries DNA; a fluorescent dye raised against RNA counts only RNA.

4Columns have a capacity, and a size cut-off you did not ask for

A silica spin column binds RNA out of guanidinium and ethanol by the same mechanism it binds DNA, and it has two limits worth knowing. The first is capacity: a standard mini column holds about 100 µg of RNA, which is roughly five million cultured cells or 15 mg of liver, and loading more returns little extra while making the washes less effective.

5Residual genomic DNA, and why it matters for anything that amplifies

Silica binds DNA as well as RNA, and even an acid-phenol prep carries a little DNA across, so every RNA prep destined for an amplification assay has a DNA problem to size up. It matters because reverse-transcription qPCR cannot tell cDNA from genomic DNA: if the amplicon sits inside a single exon, or the gene has no introns, or the target has a processed pseudogene elsewhere in the genome, the polymerase will happily amplify the contaminating DNA and report it as expression.

Disruption while frozen: Fibrous or RNase-rich tissue is ground under liquid nitrogen or bead-milled with the reagent already in the tube, so the sample never spends a moment thawed and unprotected. A piece that thaws on the bench first can lose its 28S band before the buffer is on it.
Carrier: RNase-free glycogen or linear acrylamide added before the alcohol, so that a few micrograms of RNA have something to co-precipitate with. Below about 8 µg the pellet is invisible and gets poured away; a carrier makes it visible and holds it on the tube wall, and neither carrier interferes with reverse transcription.
On-column DNase: DNase I applied to the membrane after binding and before the washes. It costs 15 minutes and no yield, and the wash that follows takes the enzyme and the magnesium away, so there is nothing left to stop. In-solution treatment afterwards is more complete but has to be stopped with EDTA or a cleanup, never with heat.

Key terms

Practise these as flashcards →

Chaotropic salt

A salt such as guanidinium thiocyanate that disrupts ordered water. At molar concentrations it lyses cells and denatures every nuclease at once, which is what protects RNA during the prep, and what has to be washed out afterwards, because it inhibits enzymes just as effectively.

Acid phenol

Phenol equilibrated to about pH 4.5. The low pH is what sends DNA into the organic phase and the interphase while RNA stays in the aqueous phase. Buffered phenol at pH 7.9 keeps both nucleic acids aqueous and belongs to DNA work.

RNase-free

Not sterile and not autoclaved: RNase A survives autoclaving. It means certified reagents and consumables, kept for RNA work only, handled with gloves. Water and plasticware are treated with DEPC or supplied certified; glassware is baked.

Minus-RT control

The same qPCR reaction with the reverse transcriptase left out. Any signal comes from DNA, so the gap in cycles between the minus-RT and the full reaction is the only direct measure of whether residual genomic DNA matters for that assay.

DNase I

An endonuclease that cuts DNA and not RNA, used to remove genomic DNA from an RNA prep. It needs divalent magnesium, which means it must not be heat-inactivated in place: heating RNA with magnesium in the tube hydrolyses the backbone.

Integrity score

A number from about 1 to 10 computed by an electrophoresis instrument from the whole size distribution of a sample, not just the ribosomal peaks. It is the standard way to say how intact RNA is, and it falls before the ribosomal bands look obviously wrong.

Small RNA cut-off

The size below which a silica membrane stops retaining RNA, set by the ethanol fraction in the binding mix. Around 35% ethanol the cut-off is near 200 nucleotides; raising the ethanol to 50–65% brings tRNA, 5S and miRNA onto the column.

A260/A230

The purity ratio that sees guanidinium, phenol and residual salt. Clean RNA sits near 2.0–2.2; well under 1.8 means something is in the tube that reverse transcriptase will notice, even when A260/A280 looks respectable.

When to use it

Measuring gene expression by RT-qPCR or RNA-seq, where integrity and residual genomic DNA decide whether the numbers mean anything.
Building a poly-A or ribo-depleted sequencing library, which needs intact RNA in a known amount: a degraded input gives a 3′-biased library however well the rest of the prep is done.
Profiling small RNA or miRNA, where the choice of chemistry decides whether the molecules of interest are in the tube at all.
Banking RNA from a tissue or cohort collection, where the sample is irreplaceable and handling at collection sets a ceiling on everything done later.

Compared with alternatives

RNA integrity check →

The next step every time. It is the only measurement that tells you whether the RNA is in one piece, and a concentration reading cannot substitute for it.

Reverse transcription →

Where the quality of this prep is cashed in: carry-over inhibits the enzyme, residual genomic DNA is indistinguishable from cDNA, and degraded RNA biases the product towards the 3′ end.

Genomic DNA extraction →

Prefer it when the target is DNA. The same guanidinium chemistry, but with neutral phenol or no phenol at all, no RNase anxiety, and an RNase A step instead of a DNase step.

DNA quantification & QC →

The quantification logic carries over: absorbance counts anything that absorbs, a dye counts what it was raised against, and neither sees integrity.

RNA-seq →

The application that sets the specification for the prep: input mass, integrity threshold and whether small RNA has to be kept.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Keeps the whole size range, from 28S down to miRNA, if the precipitation is given time in the cold, which is its main advantage over a standard column.
No binding capacity to saturate, so it scales from a needle biopsy to a gram of tissue; what limits it is the tissue per millilitre of reagent, about 30 mg, because the sample itself dilutes the chaotrope.
The interphase is the hazard: pipetting into it puts phenol and genomic DNA straight back into the sample, and phenol stops reverse transcriptase outright.
Phenol is corrosive and the reagent contains guanidinium thiocyanate, so it needs a fume hood and must never meet bleach.