Primary Author: Quoc Hoang*
Additional Authors: Brittan Pasloske Ph.D., Nathan Harris*, Jennifer Van Dinther*
*PerkinElmer Life Sciences, 2200 Warrenville Road, Downers Grove, IL 60515
Overview
- Automated protocol for one-step RT-PCR on tissue cultured cells, eliminating need for a separate RNA isolation step
- Cells are washed, lysed, DNase treated and prepared for one-step RT-PCR using the MultiPROBE Liquid handling system
- Sensitive and reproducible measurement of gene expression in cultured cells
- Compatible with real-time RT-PCR
Introduction High throughput RT-PCR analysis of RNA from cells grown in tissue culture is an expensive and multi-step process mainly because of the required up-front RNA isolation. As well, RNA isolation is generally the rate-limiting step in such experiments.
Cells-to-cDNA™ II technology (US patent pending) is designed to completely bypass RNA isolation thereby increasing the throughput potential and decreasing overall costs. With a novel cell lysis buffer, cells are lysed and RNases are inactivated in a single step yielding a cell lysate that is competent for reverse transcription.
Ambion, Inc. and PerkinElmer Life Sciences now offer an automated protocol for the Cells-to-cDNA II technology using the MultiPROBE® II HT Liquid Handling System and Gripper Integration Platform (PerkinElmer Life Sciences). This automated protocol includes cell lysis, DNase I treatment, and set-up for one-step RT-PCR, that can then be used in gel-based or quantitative (real-time) PCR assays all in a 96-well format.
Data is presented showing the quantitative analysis of RNA from cultured cells in a 96-well format demonstrating reproducibility and consistency of results using the MultiPROBE® II HT Liquid Handling System. An experiment showing the stimulation of the plasminogen activator gene (t-PA) by exposure to phorbol myristate acetate (PMA) provides an example of how this technology can be used.
Experimental - Equipment and Materials
MultiPROBE II HT EX
8-probe Varispan Pipetting
Figure 1. MultiPROBE II HT EX Running Cells-to-cDNA™II Protocol.
Gripper™ Integration Platform
- Gantry-based system with 5 axes of motion
Cells-to-cDNA™II
- Complete reverse transcription kit including 1X PBS, Cell Lysis Buffer, DNase I, and both an Armored RNA® and endogenous controls
- Cell lysates are used directly in reverse transcription eliminating the RNA isolation step
- Easily adaptable to a one-step RT-PCR procedure
Method
Cell Preparation
- HeLa S3 cells are grown overnight in 0.2 ml DME media with 10% FBS with equal number of cells in each well.
- After an overnight incubation, phorbol myristate acetate (PMA) is added to final concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM, and 0 nM in the growth media in replicates of eight. Incubate at 37°C for 24 hr.
Automated Protocol
Figure 2. Screen View Using WinPREP™ Software. Screen Left: outline of protocol using Cells-to-cDNA II. Screen Right: view of deck layout.
- Place 96-well plate and lid on the MultiPROBE® II HT Liquid Handling System on the proper deck positions.
- Remove the growth media and wash the cells with 1X PBS.
- Add 0.1 ml cell lysis buffer to each well.
- Move plate to 75°C heating tile and incubate for 10 min.
- Move plate to shaker platform and add 2 µl Lysis Buffer II to each well. Shake for 2 min.
- Add 2 µl DNase I to each well and move plate to 37°C heating tile. Incubate for 15 min while shaking.
- Move plate to 75°C heating tile and incubate for 5 min to inactivate the DNase I.
- Prepare a master mix for one-step RT-PCR.
- Aliquot 20 µl of the master mix to a 96-well PCR plate.
- Add 5 µl of lysate to each well. The plate is now ready for one-step RT-PCR.
PCR
- Setup a PCR profile for an ABI 7700 as follows:
42°C for 15 minutes (reverse transcription)
94°C for 3 minutes (denaturation)
40 Cycles: 94°C for 20 seconds
60°C for 40 seconds
Results and Analysis
Conclusion
Automating Ambion's Cells-to-cDNA II Kit using the MultiPROBE® II HT Liquid Handling System is an effective way of performing high throughput RT-PCR from cells grown in tissue culture. The total time to prepare a 96-well plate for one-step RT-PCR is one hour. This proves to be a sensitive and reproducible way to measure gene expression in cultured cells.