Quick Real-Time Analysis of Small Cell Samples

Two step real-time RT-PCR is the most popular method for quantitative analysis of gene expression for a wide variety of cell types over a broad range of sample sizes. To demonstrate the wide dynamic range of cell number that can be used with the Cells-to-cDNA II Kit in real-time RT-PCR, HeLa cells were grown to ~75% confluency in T-flasks and detached by incubation with trypsin. Cells were counted, washed once with phosphate buffered saline (PBS), and serially diluted in PBS. An aliquot (5 µl) of each dilution was added to 95 µl of Cell Lysis Buffer so that the final cell concentrations ranged from 1 to 10,000 cells/µl. The samples were lysed and DNase I treated according to the standard Cells-to-cDNA II protocol. For cDNA synthesis, 5 µl of each lysate were reverse transcribed in a 20 µl reaction. GAPDH TaqMan‚ qRT-PCR analysis was performed on 5 µl of each cDNA preparation using SuperTaq® DNA Polymerase in the ABI 7700 Sequence Detection System.

Results of these experiments are shown in Figure 1. Plotting cycle threshold (Ct) versus cell concentration for GAPDH yielded a standard curve with a correlation of 0.99. Thus, Cells-to-cDNA II yields linear results for 1 to 10,000 cells/µl in a Real-time qRT-PCR two step assay. It should also be noted that the minus-template PCR control for the GAPDH experiment was negative, indicating complete removal of genomic DNA.

Figure 1. Cells-to-cDNA™ II is Compatible with Two Step Real-time RT-PCR. Cell lysates containing 1 to 10,000 HeLa cells/µl were prepared using Cells-to-cDNA II. These lysates were subjected to two step real-time RT-PCR using primers and a TaqMan‚ probe for GAPDH. A) Amplification curve. B) The standard curve of Ct value vs. cell concentration. The correlation was 0.99

The Cells-to-cDNA II procedure is also compatible with one step real-time RT-PCR, a protocol that can greatly streamline qRT-PCR. Compatibility with one step qRT-PCR is important when automation is being considered or when a large number of samples must be tested. In this example, HeLa cells were processed as for the two step RT-PCR procedure described above to produce cell lysates containing 1 to 2,500 cells/µl. A 5 µl aliquot of each cell lysate was subjected to one step RT-PCR (25 µl final reaction volume) using GAPDH TaqMan primers. The ABI 7700 was programmed for a 42°C, 15 minute incubation for reverse transcription, followed by a standard 40 cycle PCR. The entire RT-PCR took 1 hour 40 minutes.

The results of this experiment are shown in Figure 2. A plot of the Ct value versus the cell concentration of the lysate was linear from 1 to 2,500 cells/µl for GAPDH, demonstrating the kit's utility for use in one step RT-PCR.

Figure 2. Cell Lysates are Compatible with One Step Real-time RT-PCR. HeLa cell lysates of 1 to 2,500 cells/µl were generated using the Cells-to-cDNA™ II protocol. The cell lysates were used directly in one-step RT-PCR and analyzed by real-time PCR for GAPDH using the ABI 7700. The inset is the standard curve of Ct value v. cell concentration. The correlation was 0.99.

The suitability of formalin-fixed cells as starting material for the Cells-to-cDNA II procedure was tested using replicate HeLa cell samples. After washing with PBS, one sample was incubated in PBS and the other was fixed in 1% formalin in PBS at 4°C for 1 hour. The cells were washed to remove residual formalin from the fixed cell sample, and then both samples were lysed in Cell Lysis Buffer at concentrations ranging from 4 to 2,500 cells/µl. After DNase I treatment, each lysate was used in a GAPDH two step RT-PCR. The Ct values for the fixed and unfixed cells were found to be equivalent (Figure 3). These data suggest that it is possible to fix and immunostain cell populations, sort the cells by flow cytometry, and then analyze gene expression in sorted cells using Cells-to-cDNA II.

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, frozen sections of mouse liver embedded with OCT medium (Tissue-Tek) were fixed and then stained with Hematoxylin-Eosin. Sections were cut to 5-10 µm with a cryostat. Tissue samples of 0.16, 0.36, and 0.64 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. The lysates were analyzed for GAPDH using one step real-time RT-PCR with the ABI 7700. GAPDH was detected in all of the samples, indicating that LCM sections are suitable samples for the Cells-to-cDNA II procedure.

Figure 3. Cells-to-cDNA™ II is Compatible with Fixed Cells. HeLa cells fixed with 1% formalin and then processed using the Cells-to-cDNA II procedure generated almost identical Ct values for GAPDH as untreated cells. This was true up to 2,500 cells/µl of cell lysate.

To measure changes in gene expression in high throughput mode, Cells-to-cDNA II can be adapted for use on an automated platform. The Cells-to-cDNA II procedure does not require magnets, phenol extractions, centrifugation or vacuum processing and is therefore, ideal for high throughput analysis and automation.

As an example, we replicated a previously published observation that phorbol myristate acetate (PMA) increases tissue plasminogen activator (tPA) levels in human endothelial cells (Costa, 2001). HeLa cells were seeded in a 96 well plate. PMA was added to the cells at 0, 0.1, 1, 10 and 100 nM, in replicates of eight. The cells were incubated for 24 hours at 37°C and then processed through the Cells-to-cDNA II protocol using a Packard MultiPROBE® II robot (PerkinElmer). The Packard robot was programmed to distribute 5 µl of each lysate to 96 well PCR plates containing the reagents for one step RT-PCR. Primers and TaqMan‚ probes for tPA and for 18S rRNA were added to each sample. The 18S rRNA signal was used as an internal control for generating normalized values for tPA expression levels.

As shown in Figure 4A, the signal from 18S rRNA remained essentially constant for all samples whereas the level of tPA expression rose with an increase in the PMA concentration. After normalization of the tPA signal with that of 18S (Figure 4B), the abundance of tPA mRNA was shown to increase by as much as 29 fold after PMA treatment.

A. Before normalization to 18S rRNA

B. After normalization to 18S rRNA
 
Figure 4. Automating the Cells-to-cDNA™ II Protocol for Drug Induction Studies. Analysis of tPA induction by PMA in HeLa cells using Cells-to-cDNA II and the Packard MultiPROBE® II from PerkinElmer. HeLa cells were grown in 96 well plates and incubated with different concentrations of PMA for 24 hours. Cell Lysis Buffer was added directly to the 96 well plate and then processed according to the Cells-to-cDNA II protocol. Cell lysates were used directly in one-step RT-PCR analysis of 18S rRNA and tPA. Panel A. Relative tPA values before normalization against 18S rRNA signals. Panel B. Corrected tPA values after normalization with 18S rRNA. 

Costa M, Medcalf RL (2001) Ectopic expression of the cAMP-responsive element binding protein inhibits phorbol ester-mediated induction of tissue-type plasminogen activator gene expression. Eur. J. Biochem. 268: 987-996.