Real-time PCR has become the method of choice for quantitating gene expression.

When PCR was developed, it was recognized as a powerful method to detect minute amounts of particular DNA sequences in samples. The potential for quantitation of input target was also recognized, and researchers quickly began developing strategies to harness the reaction for relative and absolute target quantitation. The introduction of real-time PCR greatly facilitated target quantitation because it measures the quantity of reaction products at each cycle, making it much easier to limit measurements to the exponential (and therefore accurate) phase of amplification. Real-time PCR is accurate over a 9-log dynamic range, and can detect small quantities of targets, even as little as a single copy in some cases.

Absolute Quantitation
Initial real-time PCR quantitation strategies relied heavily on accurate mass amounts of input template. The so-called “absolute quantitation” method involves creating a standard curve of amplification of known amounts of target, and then using the observed CT values from different known input amounts for comparison to samples containing unknown target amounts. Other researchers have simply used the same mass amount of each sample and compare the “raw” CT values from real-time PCR for the desired target; this is referred to as “mass normalization”.

Relative Quantitation
Compared to absolute quantitation, relative quantitation is significantly quicker and easier, and is thus becoming the preferred method for real-time PCR data interpretation. Relative quantitation of gene expression involves reverse transcription (RT) followed by real-time PCR of a control target (with constant, characterized expression levels) to normalize expression of unknown targets; these control targets are often called “normalizers”. (see Selecting Endogenous Controls for Real-time PCR.) With the right real-time PCR reagents and instruments, both the target-of-interest and the endogenous control can be amplified in a single “duplex” reaction, adding to the simplicity of this method.

The most common method for quantitating gene expression is to isolate RNA from the samples to be compared, quantitate the RNA by UV spectrophotometry or with a fluorescent dye such as RiboGreen® Reagent (Invitrogen), and then use equal mass amounts of RNA in real-time RT-PCR. This workflow provides good results, but isolating RNA is time-consuming, labor intensive, and provides ample opportunity for sample degradation. Furthermore, RNA quantitation is prone to error from machine or pipette miscalibration, or dilution, and because of its limited 100–fold dynamic range, these methods often require sample dilution for accurate measurement. In addition, spectrophotometry cannot be used to detect RNA from low cell inputs; usually RNA from at least 104 cells is needed for accurate quantitation. Using a fluorescent dye increases sensitivity up to 100–fold, but can be expensive.

Ambion Cells-to-CT technology enables real-time PCR of cultured cell samples without isolating RNA. Potential problems such as sample mix-up and RNA degradation are minimized, and samples are not subject to RNA quantitation errors. With Cells-to-CT kits, cell lysates are ready for reverse transcription (RT) after a two-step, 7-minute lysis procedure at room temperature that includes an option for DNase treatment to simultaneously remove genomic DNA. Samples can contain 10–100,000 cells, and prepared lysates can be used in RT reactions without quantitation of any kind. Customers sometimes ask how they can quantitate the RNA in their Cells-to-CT lysates—the answer is that it is not necessary and here we present data to support this claim (see also "Skipping RNA Quantitation: Common Concerns", in sidebar).

RNA was purified from 10–100,000 HeLa cells using the Ambion MagMax™-96 Total RNA Isolation Kit and quantitated using either a NanoDrop™ spectrophotometer or RiboGreen reagent. Spectrophotometry was not sensitive enough to measure the RNA isolated from from 10, 100, or 1,000 cells. RiboGreen Reagent could detect RNA isolated from as few as 100 cells, but the RNA concentration measured was at the limit of detection reported by the manufacturer, making quantitation less precise.

To evaluate whether determining RNA concentration is important prior to relative quantitative RT-PCR with Cells-to-CT, we compared real-time RT-PCR data for an endogenous control and a gene known to be expressed in our cell line, from either normalized mass amounts of input RNA or Cells-to-CT lysates made from 10–100,000 cells (Figure 1). Direct reverse transcription and real-time PCR from Cells-to-CT lysates gave equivalent results to quantitation by normalizing to equal mass amounts of RNA. In addition, the Cells-to-CT method is accurate over a large dynamic range, is extremely sensitive, and simplifies your real-time PCR workflow.