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Extraction, purity, PCR formats and controls

15 min

  • Choose an extraction method for a DNA, RNA, or cell-free nucleic acid target
  • Accept or reject a nucleic-acid extract using A260/A280, integrity, and amplifiability
  • Choose endpoint, real-time, digital, or RT-PCR for the target and for quantitation
  • Locate the failed step of a molecular test from where each control is added

Read the full reference

Try first

Try first

A plasma specimen arrives for a cell-free DNA assay. The only extraction protocol set up on the bench today is the laboratory's whole-blood genomic DNA method. What do you do?

The next section explains it.

The next section explains it.

Right. The next section explains why.

The next section explains it.

Get the idea

Extraction matches the target

Every extraction lyses the specimen and removes substances that inhibit or interfere with the next step. How much target it recovers depends on the matrix, where the target sits, its length, the input and the elution volume.1 A method that recovers plenty of genomic DNA from white cells can lose short cell-free DNA fragments or RNA. RNA work also controls ribonuclease exposure and adds a reverse-transcription step before amplification.1,2

Purity, integrity and amplifiability

Absorbance at 260 nm estimates total nucleic acid. The A260/A280 ratio of pure DNA is near 1.8, and pure RNA is near 2.0.3 A low ratio points to gross protein or solvent contamination. An acceptable ratio says nothing about fragment size, degradation or inhibitors. Electrophoresis shows size and degradation, and an amplification control shows whether the extract amplifies. The assay's own acceptance limits decide whether an extract is used.1

Choose the PCR format for the question

FormatWhat it measures well
End-point PCRPresence of product after cycling
Reverse-transcription PCRAn RNA target, copied to DNA first
Real-time PCRStarting quantity, read from the quantification cycle (Cq) against a standard curve
Digital PCRStarting quantity, counted as positive partitions

Reactions that start with very different amounts of target reach similar signals at the plateau. End-point signal therefore cannot rank starting quantity.2 A lower Cq means more amplifiable target, and Cq values compare only across runs with compatible efficiency, threshold and calibration.4

Each control covers the steps it passes through

  • Negative extraction control: enters before extraction and detects contamination from extraction onward.
  • No-template control: enters at reaction setup and detects contaminated reagents or setup.
  • Positive control: detects failure to find a known target, over the steps after the point where it is added.
  • Internal amplification control: travels with each specimen and detects inhibition or reaction failure.2,5

A positive control added after extraction cannot show that extraction worked. When a control fails, the failed step lies between the point where that control entered and the end of the run.5

References
  1. Clinical and Laboratory Standards Institute. Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods. 2nd ed. CLSI guideline MM13. Clinical and Laboratory Standards Institute; 2020. Accessed September 26, 2026.
  2. Kralik P, Ricchi M. A basic guide to real time PCR in microbial diagnostics: definitions, parameters, and everything. Front Microbiol. 2017;8:108. doi:10.3389/fmicb.2017.00108
  3. Powell EA, Mortensen JE. Extraction of total nucleic acids from bacterial isolates using the bioMérieux NucliSENS easyMAG total nucleic acid extractor. Ann Clin Microbiol Antimicrob. 2016;15(1):54. doi:10.1186/s12941-016-0168-7
  4. Bustin SA, Ruijter JM, van den Hoff MJB, et al. MIQE 2.0: revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines. Clin Chem. 2025;71(6):634-651. doi:10.1093/clinchem/hvaf043
  5. Clinical and Laboratory Standards Institute. Molecular Diagnostic Methods for Infectious Diseases. 3rd ed. CLSI report MM03. Clinical and Laboratory Standards Institute; 2015. Accessed September 26, 2026.

Watch one

A DNA extract from a blood specimen is ready for an assay whose target is 450 base pairs long.

  • Concentration is 42 ng/µL, above the assay's minimum input.
  • A260/A280 is 1.84.
  • Capillary electrophoresis shows most fragments below 300 base pairs, with little intact high-molecular-weight DNA.

Do you accept the extract for the assay?

  1. Read the ratio: 1.84 is near the 1.8 expected for DNA, so gross contamination is unlikely.

    The ratio is the quickest screen for gross protein or solvent contamination.

  2. Look at integrity next: most fragments are shorter than 300 base pairs.

    A clean ratio carries no information about fragment size or degradation.

  3. Compare with the target: a 450-base-pair target needs fragments at least that long, and few are present.

    The assay can amplify only a template that spans its whole target.

  4. Decide against the assay's acceptance limits: this extract fails the integrity requirement.

    Degraded input amplifies poorly and can produce a false negative.

  5. Re-extract from the specimen under the procedure, or request a new specimen if the specimen itself is degraded.

    A new extraction shows whether the degradation came from the extract or the specimen.

Reject the extract for this assay. The ratio is acceptable, and the DNA is too fragmented for a 450-base-pair target.

Your turn

Problem 1 of 3

In a polymerase chain reaction (PCR) run, a patient specimen shows no target signal and no internal control signal. The internal control was added to the specimen before extraction. The positive control added at reaction setup is detected, and the no-template control is negative. Where does this evidence place the failure?

Incorrect. The positive control used the same reagents and cycling and was detected, so amplification worked in this run.

Correct. Only the control that went through this specimen's extraction failed, which points to extraction loss or an inhibitor carried into the extract. The result is invalid. Retest the specimen from extraction or request a new collection.

Incorrect. A specimen without the target still gives an internal control signal. With that signal missing too, the run is invalid for this specimen, and a not-detected result cannot be reported.

Hint
  1. Find which controls worked, and the steps each of them went through.
  2. The positive control added at setup went through amplification and detection only.
  3. Only one control in this reaction went through this specimen's extraction.

Review Controls that localize failure

Problem 2 of 3

A test will report a viral load in copies/mL so that results from month to month can be compared. Which format fits?

A gel band is read after the reaction has reached its plateau. Reactions that started with very different amounts end with similar bands, so the band cannot give copies/mL.

Used endpoint plateau signal to estimate starting quantity

Once reagents run out, reactions that started with very different amounts of target reach similar plateau signals, so an endpoint reading cannot rank starting quantity. Real-time quantification cycle (Cq) values read against a standard curve, or digital partition counts, measure starting quantity.

Cq falls earlier as starting target rises. Read against a calibrated standard curve, it gives a starting quantity that can be reported in copies/mL.

Nested PCR adds a second round of amplification and carries a high risk of carryover contamination. Its end product does not measure the starting quantity.

Hint
  1. The question asks how much target was present at the start.
  2. Think about what happens to the signal once reagents run out.

Review Polymerase chain reaction

Problem 3 of 3

A DNA extract has a concentration of 60 ng/µL and an A260/A280 ratio of 1.52. Electrophoresis shows mostly intact high-molecular-weight DNA. What does the ratio tell you?

Intact DNA shows the fragments are long. The low ratio is a separate finding, and it points to contamination that can interfere with the assay.

Pure RNA gives a ratio near 2.0, higher than DNA. RNA in the extract would push the ratio up.

At 60 ng/µL the concentration is well within a readable range. The low ratio is a real finding here.

Pure DNA reads near 1.8. A ratio of 1.52 points to protein or solvent carried over from extraction, which can inhibit the reaction.

Review Quantity, purity, and integrity

Use it

  • A reverse-transcription real-time PCR run for a respiratory RNA virus holds 22 patient swabs.
  • The negative extraction control went through extraction with the patients and shows the viral target at Cq 35.1.
  • The no-template control, added at reaction setup, shows no amplification.
  • The positive control is detected at its expected Cq.
  • Every internal control is detected in range.
  • Three patient swabs show the viral target at Cq 34.6 to 36.8.
  • The run's patient results are waiting for release.
Decision 1 of 3

Where does the evidence place the contamination?

The no-template control used the same master mix and stayed negative. Contaminated reagents would have shown target there too.

The negative extraction control picked up target, and the no-template control added at setup did not. The contamination entered between the start of extraction and reaction setup.

Any target in a negative control is a failed control, whatever its Cq. Late Cq values in three patients are the pattern low-level contamination produces.

Review Controls that localize failure

Decision 2 of 3

What do you do with the run's patient results?

The failed negative control invalidates the run under the procedure. Contamination in extraction can affect every specimen in the batch.

A comment does not make a contaminated positive belong to the patient. Those results could be acted on as true infections.

The run is invalid. Cleaning removes the source, and a repeat from extraction with new controls shows whether the three positives are real.

Review Controls that localize failure

Decision 3 of 3

The RNA extraction instrument will be busy for 2 hours. A colleague offers to re-extract the swabs now on the whole-blood genomic DNA protocol on the second instrument. What do you do?

A genomic DNA method is not validated for RNA from swabs and gives no ribonuclease control. Poor RNA recovery reads as a negative result for virus that was present.

Used a genomic DNA extraction for an RNA or cell-free target

A method that recovers abundant genomic DNA can recover RNA or short cell-free fragments poorly, and RNA also needs ribonuclease control and a reverse-transcription step. The low recovery then reads as a negative result for a target that was present.

The assay's performance was established with its own extraction. The 2-hour wait keeps the repeat results reliable.

Extra enzyme cannot convert RNA the extraction failed to recover. It also moves the reaction outside its validated conditions.

Review Extraction and purification

The clue that settles this case is which negative control picked up the target. The negative extraction control went through extraction and turned positive. The no-template control entered later and stayed clean. That places the contamination in extraction, so the whole run is repeated from extraction with the method the assay was validated with.

Keep

Sources checked