| Synthesize cDNA Directly From Limiting Samples
No RNA Isolation Required The Cells-to-cDNA™ II Kit has an incredibly wide dynamic range, making it compatible with tissue culture cells, and very small samples such as Laser Capture Microdissected samples and FACS sorted cells. Use Laser Capture Microdissection (LCM) Cell Samples Directly for RT-PCR LCM is a powerful technique for isolating small numbers of specific cells for gene expression analysis. To demonstrate the utility of Cells-to-cDNA II for analysis of LCM samples, 5-10 µm frozen sections of mouse kidney embedded with OCT medium (Tissue-Tek) were fixed and stained with Hematoxylin and Eosin. Tissue samples of 0.04, 0.16, 0.36, and 1 mm2 were captured by LCM using an Arcturus PixCell II system. The plastic layer on the LCM cap holding the sections was transferred into 100 µl of Cell Lysis Buffer and subjected to the Cells-to-cDNA II procedure. Samples were analyzed for GAPDH expression levels using one step real-time RT-PCR. Results of these experiments are shown in Figure 2, below. Plotting cycle threshold (Ct)
versus cell concentration for GAPDH yielded a standard curve with a correlation coefficient of 0.95. Thus, Cells-to-cDNA II is ideal for generating cDNA for real-time RT-PCR analysis of LCM sections. | |
Working with Limiting Samples
As cell separation techniques become increasingly sophisticated, an increasing number of researchers are working with small subpopulations of cells rather than with whole tissue sections or heterogeneous cell populations. Needle biopsies and cytological smears are no longer the smallest samples around. Laser Capture Microdissection (LCM) enables the isolation of pure cell populations from fixed sections of tissues or cells. Similarly, fluorescence activated cell sorting (FACS) is a powerful tool for sorting suspended cells by biochemical or molecular markers. Both LCM and FACS enable the isolation of pure populations of cells, down to a single cell, for gene expression analysis. While there are obvious advantages to studying the gene expression of pure cell populations, it also presents many challenges. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) is commonly used for gene expression analysis on small numbers of cells, and requires RNA as a template. RNA isolation from small samples is problematic because of sample loss during handling and variability in yield. No RNA Isolation Required
The Cells-to-cDNA II Kit enables quantitative gene expression analysis directly from very small samples, such as those recovered from LCM sections and FACS. Removing the requirement for RNA isolation enables researchers to work with a limited number of cells and still obtain reproducible, robust results (Figure 2 and "Cells-to-cDNA II using FACS Sorted Cells". Cells-to-cDNA II employs a unique Cell Lysis Buffer that simultaneously disrupts cells while inactivating RNases, yet is compatible with DNase I digestion and cDNA synthesis. The procedure (Figure 1) begins with mixing your LCM or cell sample with Lysis Buffer and briefly heating to inactivate cellular RNases. This effectively "freezes" the RNA expression pattern of the sample. After a DNase I digestion and a DNase I heat inactivation step, the treated lysate may be used directly as a template for either reverse transcription or one-step RT-PCR experiments. What You Get
The Cells-to-cDNA II Kit is available in both 40 and 100 reaction sizes and comes complete with all of the reagents necessary to release and stabilize cellular RNA, remove contaminating DNA, and synthesize cDNA for any downstream application. | | Figure 1. Cells-to-cDNA II Procedure. | | | Figure 2. Direct cDNA Synthesis and Real-time Analysis on LCM Sections. Cell lysates from LCM sections of 0.04, 0.16, 0.36, and 1 mm2 were prepared using Cells-to-cDNA II. These lysates were subjected to one step real-time RT-PCR using primers and a TaqMan® probe for GAPDH. A. Amplification curve. B. The standard curve of Ct value vs. starting quantity. | |