Occasionally, one is faced with the prospect of performing RT-PCR simultaneously on numerous samples. In my case, this occurred following the retroviral transfection of cultured lung cancer cells with an shRNA targeted against a specific transcript. Since the entire puromycin-resistant population of cells showed no difference in the level of the mRNA of interest, I needed to expand cultures of individual cells in order to identify the clones that carried the knockdown. My first instinct was to locate an RNA purification method applicable to a 96-well plate. This would have been followed by an equally high-throughput cDNA synthesis reaction, and then PCR. During my search I came across a product that, supposedly, would allow cDNA synthesis directly from cell lysates without first performing an RNA isolation. The product is Ambion’s Cells-to-cDNA™ Kit II (now sold by Applied Biosystems, Life Technologies).
The protocol is very simple. Cultured cells are washed once with PBS and then lysed with the lysis buffer provided with the kit. To discourage RNase activity, the cells are lysed on ice with ice-cold lysis buffer. The cells lyse instantly and are immediately incubated at 75°C. After a 10 min incubation, the lysates are placed on ice and DNase I (provided with the kit) is added. Following a 15 min incubation at 37°C, the DNase is inactivated by heating to 75°C for 5 min. The lysates are then ready for cDNA synthesis, or a one-step RT-PCR procedure.
The procedure is quite flexible in that the cells can be lysed in a multi-well plate and then transferred to a PCR plate for heating; or harvested from a plate, transferred to the PCR plate, and then lysed. Since the Cells-to-cDNA™ protocol calls for incubations at several different temperatures (75°C, 37°C, and 42°C including the cDNA synthesis), it is clearly advantageous to use a PCR machine for heating.
In a pilot experiment, I prepared lysates from 390, 780, 1560, 3125, 6250, 12500, or 25000 cells. I then used 5 µl of each lysate in 20 µl cDNA synthesis reactions using either MMLV-RT (included with the kit) or Transcriptor-RT (from Roche). The MMLV-RT reaction was carried out at 42°C and the Transcriptor-RT at 55°C. I then used 2 µl of each cDNA in 20 µl PCR reactions containing the primers that I was planning to use for the knockdown screen. These primers generate an amplicon of ~300 bp. The figure to the left shows the results.
The PCR reactions in Panel A used MMLV-RT-transcribed cDNA and Panel B used Transcriptor-RT-transcribed cDNA. The gray triangle shows the number of cells lysed, starting with 390 cells and increasing 2-fold up to 25000 cells. The entire 20 µl PCR reaction for each condition was loaded into each well of the 1.7 % agarose TAE gel. As can be seen in the figure, clear bands are present at the expected size for all cell numbers. The PCR reactions employing MMLV-RT cDNA show some higher MW smearing that is not present with the Transcriptor-RT cDNA. This may be a reflection of the different incubation temperatures used with MMLV-RT or Transcriptor-RT (42°C vs. 55°C, respectively). The lanes marked (-) and * signify negative (minus RT) and positive (cDNA made from RNA isolated from the same cells using a conventional method) controls.
Overall, the Cells-to-cDNA™ Kit II performed exceptionally well. The protocol was simple and resulted in high quality cDNA, as evidenced by the positive PCR results achieved with less than 400 cells. Since the intensities of the bands in the gel are clearly sensitive to cell number, the kit may be more applicable to exercises that require only a yes or no answer as opposed to those that require an accurate measure of the quantity of a particular mRNA species. For example, the kit should permit the identification of cell clones demonstrating successful shRNA knockdown, but may not suffice for experiments designed to identify mRNA abundance differences of just a few-fold. For the latter, subtle differences in mRNA level may just as well be due to differences in starting cell number as to biology.
Associate Research Professor
Department of Radiology
Duke University Medical Center