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Cells-to-Signal™ Kit
Monitoring the Effects of Drug Treatment
on mRNA Expression
The Cells-to-Signal Kit™ (patent pending)
uses a chemical lysis method to create cell lysates in less than
5 minutes at room temperature. These cell lysates can be used
directly for qRT-PCR when PCR primers are designed to span intron-exon
boundaries, thus bypassing RNA isolation altogether. The Cells-to-Signal
procedure is highly reproducible, compatible with a wide variety
of cell types, and has been optimized for high throughput analyses.
Here we present an example of how the Cells-to-Signal Kit was
used in our laboratories at Ambion to monitor the effects of
phorbol myristate acetate (PMA) treatment on the expression of
plasminogen activator mRNA.
PMA Induction of Plasminogen Activator
HeLa cells grown in 96 well plates were treated
with phorbol myristate acetate (PMA), a potent tumor promoter,
at final concentrations of 100, 10, 1, 0.1, and 0 nM, for 24
hours. Cells were then washed with PBS and processed with the
Cells-to-Signal Kit. This involved simply lysing the cells for
5 minutes at room temperature with 100 µl of Cells-to-Signal
Lysis Buffer. One-step real-time RT-PCR (10 µl reaction)
was performed on 2 µl of the cell lysate using primers
and a TaqMan® probe targeting tissue plasminogen activator
(t-PA).
t-PA expression was detected in duplicate samples
by real-time PCR at all PMA concentrations (Figure 1). As expected, the
level of t-PA mRNA increased when the cells were grown in the
presence of PMA [1].
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Figure 1. PMA Induction of Plasminogen Activator (t-PA). HeLa cells were seeded in DME media with 10% FBS in a 96 well tissue culture plate at 3,000 cells/well and grown overnight. PMA was added to the growth medium at the designated final concentrations and the cells were incubated at 37°C for an additional 24 hr. Cell lysate (2 µl) was used for one-step, real-time RT-PCR (10 µl reaction) with primers and a TaqMan® probe targeting tissue plasminogen activator (t-PA). 18S rRNA was similarly targeted and used as a normalization control for target input. |
These results demonstrate that Cells-to-Signal
can be used to quickly examine multiple cell culture samples
for specific gene expression. Cells-to-Signal Kits contain reagents
for 30 or 100 reverse transcription reactions, including M-MLV
RT, oligo(dT)18 primers, and random decamer primers. Up to 5
x 104 cells per reaction can be used for the lysis
step. Various cell lines, including BJ, HeLa, HeLa S3, MCF-7,
K562, SKNAS, and NHDF-neo have been successfully used with the
Cells-to-Signal Kit. The kit also contains complementary primers
and a positive control RNA for optimizing the procedure.
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