RT-qPCR for relative expression

RT-qPCR for relative expression asks whether a transcript is more or less abundant in one sample than in another, after you account for how much RNA you actually measured. It does not, by itself, count molecules per cell. That stronger claim needs a calibration with known copies. This page explains the relative comparison: reverse transcription, the quantification cycle, reference transcripts, and the controls that make a fold change meaningful. The surrounding design, from harvest to the choice of assay, is in from cells to a gene expression result.

The comparison you are really making

You pick a calibrator sample. You measure the target transcript and one or more reference transcripts in every sample, including the calibrator. The usual arithmetic subtracts the reference cycle from the target cycle, then subtracts the calibrator's value, then converts the cycle difference into a fold change on a log scale. The familiar power-of-two conversion assumes that each cycle doubles the product. If the reactions are less efficient, that conversion overstates the fold. Measure efficiency. Do not borrow it from a methods sentence in another paper.

The MIQE guidelines set out what a published real-time experiment should report, including efficiency, the reference-gene justification, and negative controls. They are a reporting discipline. They are not a master-mix recipe.

What happens in the tube

Reverse transcription copies RNA into complementary DNA. The priming strategy changes which molecules you see. Oligo(dT) priming follows polyadenylated tails and will under-represent broken RNA and many non-polyadenylated transcripts. Random primers copy more broadly and can also copy ribosomal RNA. Gene-specific primers are narrow and useful when one assay is the whole experiment. The enzyme class, the temperature, and the time belong to the enzyme you have. Write them down from that label.

The quantitative PCR step watches the product every cycle. A dye that binds double-stranded DNA, in the SYBR Green family, fluoresces whenever any duplex forms. It is simple, and it will also score primer-dimer and any off-target amplicon. A hydrolysis probe fluoresces when a particular internal sequence is copied. It is more specific and still fails if the probe design is wrong or the well is inhibited. The quantification cycle, often called Cq or Ct, is the cycle at which the signal crosses a threshold you define. A lower cycle means more starting template only when the efficiencies match and the threshold rule is shared.

Primers carry the specificity. Design them on a stated transcript from a browser such as Ensembl, and check what else they might bind with NCBI Primer-BLAST. Where the gene has introns, an exon-exon junction primer pair lets genomic DNA fail to amplify at the same size. That design is a help, not a waiver of the no-reverse-transcription control.

Reagent and plate classes

A reverse transcriptase, an RNase inhibitor, dNTPs and a buffer are the functional reverse-transcription mix. A hot-start polymerase and a fluorescent chemistry are the functional qPCR mix. Some suppliers combine them in one step. One-step and two-step formats answer different practical needs: two-step lets you archive cDNA and run many genes from one transcription; one-step reduces handling and also commits the RNA to a single assay. Enzyme catalogues such as the NEB product pages illustrate those classes. Match the enzyme you actually open.

Plasticware is optical. White plates and clear plates change the signal the instrument collects. Sealing film must be optical and flat. A fingerprint, a crease, or a poorly seated seal becomes a well that looks like biology. The photograph of a sealed plate is a reminder that the seal is a reagent.

Reference genes are not a universal set. A housekeeping gene is simply a transcript that has been stable in somebody's conditions. Test candidate references across your own treatments. If the treatment changes the reference, every target you normalise to it will shift, including targets that did not change.

Branch points when a control fails

No-template controls contain every reagent except nucleic acid. Amplification there means contamination or a primer artefact. The plate's sample wells do not get a fold change that day. No-reverse-transcription controls contain RNA and the qPCR reagents without the reverse transcriptase. Amplification there means genomic DNA or a contaminated enzyme. Redesign the primers or repeat the DNase step. Do not subtract a little and carry on unless you have shown the contribution is negligible in a way you can document.

A dilution series of a representative cDNA gives you efficiency. Plot Cq against the log of the template amount. A reaction that doubles each cycle has a slope near minus 3.3. Laboratories often accept a window around that ideal and still report the measured efficiency. If the target and the reference differ sharply, the simple fold-change formula is the wrong model. Use a method that takes the measured efficiencies into account, or fix the assays.

Melt curves, for dye chemistries, should show one product at the temperature you expect. A second peak is a second product. A probe assay skips the melt and still needs evidence that the amplicon is the one you designed, at least when the assay is new.

Where RNA-seq takes over

RNA-seq is the better tool when you do not yet know which transcripts matter, or when you suspect the references themselves are moving and you need a transcriptome-wide view. It is a different measurement, with library bias and a different statistical model. A single RT-qPCR assay can still be the right confirmation of a candidate that came from a sequencing table. The two methods agreeing is stronger than either one quoted alone. They will not agree to the decimal if the biases differ. Look for the same direction and a design that can support it.

Failure modes that mimic a fold change

Inhibition makes Cq later. A dirty sample looks weakly expressed. Dilute the cDNA or spike a control template into the sample matrix. If the spike is also late, you are measuring dirt. Saturation and a threshold set inside noise make early cycles meaningless. Set the threshold in the exponential region and use the same rule for every well in the comparison.

Pipetting error between the target plate and the reference plate becomes a false fold. Technical replicates catch gross misses. They do not create biological replication. If you have one culture, you have one biological result, however many wells you filled.

Safety, climate and the enquiry

The everyday hazards are the RNA extraction chemicals upstream, hot blocks, and any infectious source material. Follow institutional biosafety rules for the cells. This assay does not diagnose infection or disease.

In humid weather, plates that wait unsealed concentrate or pick up condensation. Seal them and spin droplets down before the run. A cycler that rebooted after a power cut needs a completed run with controls before you trust a half-written file. Enzymes follow their own storage labels. A warm shipping day is a reason to check a known positive RNA, not a reason to adjust the threshold until a fold change appears.

For a sourcing question, name the organism, the transcripts, one-step or two-step, dye or probe, the reference strategy, and whether you also need a transcriptome-wide method. The nucleic acid analysis pathway places the assay in that wider route. The mRNA sequencing reference and the differential expression analysis reference are independent pages for a conversation about RNA-seq when the gene list outgrows qPCR. Use the quote request to ask whether a quotation is possible. Those pages do not say that EVRINTH operates the assay or holds a particular master mix.