RT-PCR Without Prior RNA Isolation

RT-PCR Without Prior RNA Isolation

Isolating RNA from small numbers of cells for quantitative RT-PCR is a difficult undertaking because sample loss can occur during any or all of these steps: cell lysis, RNA extraction, or RNA recovery. The Cells-to-cDNA™ Kit solves these problems by integrating RNase inactivation and DNase I treatment into a single Cell Lysis Buffer, therefore eliminating RNA isolation completely. The Cells-to-cDNA cell lysis procedure is fully compatible with RT-PCR techniques so that the cell lysates can be used directly in gel-based, one-step RT-PCR as well as real-time RT-PCR experiments.

Go Directly from Cells to cDNA in Less Than 2 Hours

The first step in the Cells-to-cDNA procedure involves washing the cells to remove proteins and other potential inhibitors from the tissue culture medium (see Figure 1). The washed cells are resuspended in Cell Lysis Buffer and heated to simultaneously rupture the cell membranes, release RNA and inactivate RNases. This is followed by DNase I treatment of the lysate and heating a second time to inactivate the DNase I. Traditional RNA isolation kits use strong denaturants, which if carried over into enzymatic reactions would inhibit or even inactivate the enzymes. The Cells-to-cDNA Cell Lysis Buffer has been specifically formulated so that the resulting lysate can be used directly as a template for cDNA synthesis. A fraction of the lysate is added to a reverse transcription reaction (reagents included in the kit). Cell Lysis, DNase treatment, and first-strand cDNA synthesis is completed and ready for PCR applications in just 2 hr.

Figure 1. Cells-to-cDNA Procedure.

The Cells-to-cDNA protocol is optimized for processing a wide range of cell numbers. Samples of just a single cell up to 10,000 cells (per 100 µl of Cell Lysis Buffer) can be used. Adherent cells grown in 96-well plates, can also be used with the kit. The kit is ideal for analyzing differentially treated samples (e.g. for screening drugs, for time course experiments), or for amplifying a sequence for cloning.

Compatible with Multiple Cell Lines for Diverse Applications

The Cells-to-cDNA Cell Lysis buffer is designed to work with a wide range of cell types. Table 1 shows a list of mammalian cell types from multiple species that have been used successfully with the Cells-to-cDNA Kit. Figure 2 demonstrates end point RT-PCR amplification of cyclophilin from several mouse and human cell lines processed using the Cells-to-cDNA Kit. Figures 3a and 3b show real-time RT-PCR data from a titration of HeLa cells subjected to the Cells-to-cDNA procedure.

Figure 2. Amplification of Cyclophilin From Different Cell Lines. Lysates were made from different cell lines by disrupting cultures in Cells-to-cDNA™ Lysis Buffer at a concentration of 100 cells/µl. 5 µl of the cell lysate was used as template for the RT reaction and 5 µl of the first-strand cDNA was used for PCR. After 40 cycles of PCR, 20 µl of the reactions were electrophoresed on a 2% agarose gel.

Figure 3a. Amplification of GAPDH From HeLa Cells By One-step Real-time RT-PCR. HeLa cell lysates were made by diluting cells to 25, 50, 100, 200, 400 and 800 cells/µl with the Cells-to-cDNA Cell Lysis Buffer. 5 µl of each lysate was used as a template for 25 µl one-step RT-PCR reactions using the Applied Biosystems TaqMan® One-step RT-PCR Kit. The GAPDH PCR product was detected with a JOE-(ABI) labeled GAPDH TaqMan probe.

Figure 3b. Standard Curve. Standard curve derived from the amplification profiles shown in Figure 3a. X-axis, cell number; Y-axis, CT value.

The Cells-to-cDNA Kits come with all the necessary reagents for 40 or 100 reverse transcription reactions, including reverse transcriptase, RNase Inhibitor, and both oligo(dT) and random primers. An RNase-resistant RNA (Armored RNA®) provided with the kit can be used as a positive control for monitoring inactivation of ribonuclease contamination and to assess reverse transcription efficiency.