11.2 RT-PCR, qPCR, and Blotting

Core Explanation

Measuring gene expression — at the RNA and protein levels — is central to cell biology. This topic covers three categories of techniques: reverse transcription PCR (RT-PCR), which detects RNA transcripts; quantitative PCR (qPCR), which measures transcript abundance in real time; and blotting methods (Southern, Northern, Western), which detect specific DNA, RNA, or protein molecules within complex mixtures after separation by size. Understanding the distinctions among these techniques and their proper controls is essential for interpreting the molecular biology literature.

RT-PCR, qPCR, and RT-qPCR: Clearing Up the Names

The nomenclature is a persistent source of confusion. The terms differ in both the process and the purpose:

Critical distinction: "RT" in RT-PCR stands for reverse transcription, not real-time. "Real-time" refers to the monitoring of amplification as it occurs (qPCR), not to the speed of the reaction.

Reverse Transcription (RT)

Reverse transcriptase (from retroviruses, e.g., Moloney murine leukemia virus, M-MLV) synthesizes complementary DNA (cDNA) from an RNA template. Priming strategies include:

• Oligo(dT) primers: Anneal to poly-A tails of eukaryotic mRNA — enriches for mRNA over rRNA/tRNA.
• Random hexamers: Prime anywhere along the RNA — captures non-polyadenylated RNAs but dilutes signal from specific transcripts.
• Gene-specific primers: Maximum specificity for a single target.

The resulting cDNA serves as template for subsequent PCR or qPCR.

qPCR: Real-Time Detection

In qPCR, amplification is monitored in real time by measuring fluorescence at each cycle. The key metric is the quantification cycle (Cq), also called Ct (threshold cycle): the cycle number at which fluorescence rises above a defined threshold. Cq is inversely proportional to the log of the starting template concentration — a difference of 1 Cq represents approximately a 2-fold difference in template (assuming 100% amplification efficiency).

Fluorescent Detection Systems

Quantification Strategies

Absolute quantification: Compare Cq values to a standard curve generated from known concentrations of the target (e.g., serially diluted plasmid DNA). Results are expressed as copy number.

Relative quantification: Compare Cq values of the target gene to a reference gene (housekeeping gene) within the same sample. The ΔΔCq method is most common:

1. ΔCq = Cq(target) − Cq(reference) for each sample.
2. ΔΔCq = ΔCq(treated) − ΔCq(control).
3. Fold change = 2^(−ΔΔCq).

Critical caveat: qPCR is not inherently "quantitative." It becomes quantitative only when accompanied by proper standards (absolute) or validated normalization (relative). Without these, you have a real-time PCR that measures cycles, not meaningful quantities. Housekeeping gene stability must be demonstrated empirically — no gene is universally constant across all conditions.

Blotting Techniques: An Overview

Blotting transfers biomolecules from a separation matrix (gel) onto a membrane, where they are immobilized and detected with specific probes.

Named after Edwin Southern (Southern blot, 1975); Northern and Western are geographical puns on his name, not his inventions.

General Workflow

1. Separation: Molecules separated by size in a gel matrix.
2. Transfer: Molecules transferred (blotted) onto a nitrocellulose or PVDF membrane by capillary action, vacuum, or electroblotting.
3. Blocking: Membrane incubated with non-specific protein (BSA, non-fat milk) to saturate non-specific binding sites.
4. Probing: Specific detection with a labeled nucleic acid probe (Southern/Northern) or primary antibody (Western).
5. Detection: Visualization by autoradiography, chemiluminescence, or fluorescence.

Southern Blot (DNA)

Restriction-digested genomic DNA is separated on an agarose gel, denatured to single strands, transferred to a membrane, and hybridized with a labeled probe. Used historically for gene mapping, identifying restriction fragment length polymorphisms (RFLPs), and detecting gene rearrangements. Largely superseded by PCR and sequencing for most applications but remains important in certain diagnostic settings (e.g., triplet repeat disorders).

Northern Blot (RNA)

Total or poly-A-selected RNA is separated under denaturing conditions (formaldehyde or glyoxal to prevent secondary structure) and hybridized with a labeled probe. Northern blots reveal transcript size (critical for detecting alternative splicing or degradation products) and abundance. RNA integrity is paramount — RNases are ubiquitous and resilient; all reagents and glassware must be RNase-free.

Western Blot (Protein)

Proteins are separated by SDS-PAGE (see below), transferred to a membrane, and detected with antibodies.

SDS-PAGE: The Separation Engine for Western Blotting

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins by molecular weight.

The Role of SDS

Sodium dodecyl sulfate (SDS) is an anionic detergent that performs two essential functions:

1. Denaturation: SDS disrupts non-covalent interactions (hydrogen bonds, hydrophobic interactions, ionic bonds), unfolding proteins into linear polypeptide chains. Reducing agents (β-mercaptoethanol or DTT) cleave disulfide bonds.

1. Charge normalization: SDS binds to polypeptides at a constant ratio (~1.4 g SDS per gram of protein), imparting a uniform negative charge density. The intrinsic charge of the protein becomes negligible — all proteins migrate toward the anode (+) with mobility determined almost exclusively by their molecular weight.

Without SDS, proteins would separate based on their native charge, shape, and size — a far less predictable outcome. With SDS, a protein of 50 kDa runs at approximately the same position regardless of its amino acid composition.

The Gel System

• Stacking gel (low % acrylamide, pH 6.8): Large pores; proteins concentrate into a thin band before entering the resolving gel.
• Resolving gel (higher % acrylamide, pH 8.8): Smaller pores sieve proteins by size. Higher acrylamide percentages resolve smaller proteins.

Readout

• qPCR: Amplification curves, Cq values, melt curves (for SYBR Green — a single sharp peak indicates a single product; multiple peaks indicate non-specific amplification).
• Southern/Northern: Band(s) at expected size(s) on autoradiograph.
• Western: Band(s) at expected molecular weight on film or digital imager, compared to a protein ladder.

Controls

Strengths

• qPCR is highly sensitive (detects <10 copies), has a wide dynamic range (7–8 orders of magnitude), and is relatively fast (<2 hours).
• Western blotting confirms protein presence and apparent molecular weight, detecting isoforms, cleavage products, and post-translational modifications that RNA-level methods miss.
• Northern blotting remains the gold standard for confirming transcript size and detecting splice variants, complementary to the higher-throughput RNA-Seq.

Limitations

• RNA abundance ≠ protein abundance: Transcript levels explain only ~40% of the variance in protein levels (Schwanhäusser et al., 2011; Vogel & Marcotte, 2012). Translation rates, protein degradation, and post-translational regulation all intervene.
• qPCR normalization pitfalls: Housekeeping genes are not universally stable. GAPDH changes with hypoxia; β-actin changes with cytoskeletal reorganization. Normalization to multiple validated reference genes is best practice.
• Antibody quality: Western blots are only as good as the antibodies used. Many commercial antibodies are poorly validated. A band at the expected molecular weight is not proof of specificity — knockdown/knockout controls are the gold standard.
• Southern/Northern sensitivity: Both are far less sensitive than PCR-based methods and require micrograms of DNA or RNA.

Common Interpretation Errors

• "My qPCR shows 2-fold upregulation, so protein levels must also be 2-fold." mRNA and protein abundance often correlate poorly. Translation efficiency, protein half-life, and post-translational regulation can produce protein levels that are unchanged, or even changed in the opposite direction.
• "I see a band on my Western at the expected size, so it's my protein." Cross-reactivity is common. A band at ~50 kDa could be a different protein. Knockdown or knockout samples should eliminate the band if the antibody is specific.
• "Higher Cq always means less template." Only when amplification efficiency is equal across samples. PCR inhibitors in the sample, differences in cDNA synthesis efficiency, or degraded RNA can shift Cq values without reflecting true expression differences.
• "I used GAPDH for normalization, so my data is solid." No housekeeping gene is universally stable. The MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) require validation of reference gene stability under your specific experimental conditions.

Quick Questions

Q1: A researcher reports "RT-PCR analysis showed expression of Gene X." They used SYBR Green detection on a qPCR machine. What clarification would you request?

Q2: You run a Western blot and detect a band at the expected size for your protein of interest in all lanes, including the negative control (HEK293 cells that do not express your gene). What are possible explanations?

Q3: What distinguishes a Northern blot from an RT-qPCR experiment in terms of the information obtained?