It is simple and robust, and offers benefits such as a 9-log dynamic range, rapid results, sensitivity, specificity, and reproducibility that make other methods obsolete. Traditionally, the first steps in real-time PCR are to isolate and quantitate RNA. However, these steps can require significant time and hands-on manipulation, and risk sample loss, especially from small samples. The Ambion® Cells-to-CT™ Kits provide a simple and rapid method for preparing cell lysates that can be used directly in gene expression analysis (see Figure 2). Using the Cells-to-CT methodology, relative gene expression is measured by real-time PCR directly from cell lysates, eliminating the need to purify and quantitate RNA prior to amplification. Here, we demonstrate that relative gene expression data obtained from real-time PCR using the Cells-to-CT workflow is equivalent to that obtained using purified RNA quantitated by spectrophotometry prior to real-time PCR, but with significantly less time and effort required and greater sensitivity.
Real-Time PCR for Target 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”.
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.
Simplify Relative Quantitation Using Ambion® Cells-to-CT™ Methodology
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 Concentrations from Small Samples Cannot be Measured Using Spectrophotometry or RiboGreen Reagent
Comparable Gene Expression Results from Known RNA Mass Amounts or Cells-to-CT Lysates
Figure 1. Equivalent Gene Expression Results With or Without Quantitating RNA Prior to Real-Time PCR. Panel A. Real-Time PCR Results from RNA Mass Normalization. Using the RNA concentrations measured with RiboGreen Reagent, equal mass amounts (383 pg) of RNA isolated from the indicated number of cells was reverse transcribed and the cDNA was used in duplicate real-time PCRs using TaqMan® Gene Expression Assays for ACTB (assay ID#HS03023889_g1) and LDHA (assay ID#HS00855332_g1) and TaqMan Gene Expression Master Mix. The observed CTs are equivalent, demonstrating that equal mass amounts of RNA in RT-PCR produce equivalent real-time PCR results. Panel B. Real-Time PCR Results from TaqMan Cells-to-CT™ Lysates. The indicated numbers of cells were processed using the TaqMan Gene Expression Cells-to-CT Kit and lysates were used directly in duplicate real-time PCRs. The results demonstrate that lysis, RT and real-time PCR reactions are linear over 10–100,000 input cells, and that adding a specific amount of RNA into the RT reaction is unnecessary. Panel C. Equivalent ΔCT Results. The indicated numbers of cells were processed either using the TaqMan Gene Expression Cells-to-CT Kit (orange line) or for RNA purification (green line). ΔCT values were calculated by subtracting the ACTB CT values from the LDHA CT values.
Figure 2 (sidebar). 9-log Dynamic Range of Real-Time PCR. Reverse transcription reactions from mouse NIH/3T3 cell lysate (green) or purified RNA (blue) were used for real-time PCR. 9-logs of template were efficiently detected in the presence of Cells-to-CT lysate.