3.2. DirectCell™ Oligo-dT Capture Protocol

DirectCell™ Oligo-dT Capture Protocol

This modification to conventional DriverMap protocol is designed to perform high-throughput expression profiling directly from cells grown in or sorted into 96- or 384-well plates. The DriverMap protocol can then be run directly in these same plates without transfer. The protocol allows you to streamline the mRNA purification, cDNA synthesis, and multiplex PCR steps in a single multiwell plate without any intermediate purification steps. In the first steps, the cells cultivated in a multiwell plate are lysed and transferred to an oligo-dT plate (TurboCapture 96 or 384 multi-well plates from Qiagen). The mRNA is then allowed to hybridize to the immobilized oligo dT, the cell lysate with DNA, rRNA, and other impurities are washed away, and the isolated mRNA is used directly in the cDNA synthesis step.

The procedure below has been run with a few common cell lines with 1 to several thousand cells per well. Some steps, such as lysis time, may need to be optimized for particular cell types.

Step 1: Cell Lysis

Note: For suspension cells, spin down the cells at 1,000g for 5 min before starting the protocol.

1. Remove media from attached cells grown in multiwell plate (e.g. 5-10 × 10³ cells/96 plate well or 1-2 × 10³ cells/384 plate well).

2. Add 25-ul per well (96-well plate) or 10-ul per well (384-well plate) of TCL lysis buffer (with 1% (v/v) beta-mercaptoethanol) to lyse the cells. Incubate the plate for 5 min at room temperature.

Seal the plate and store at -20°C. A freeze/thaw cycle can help optimize cell lysis and RNA yield. Keep frozen until ready to proceed with the binding protocol.

Step 2. Binding mRNA to oligo-dT plates (TurboCapture plates)

1. Transfer 10-ul of cell lysate from each well of lysed cells to a 96- or 384-well TurboCapture plate.

2. Incubate the TurboCapture plate at room temperature for 60 min in orbital shaker (100-300 rpm).

3. Wash the TurboCapture plate three times using 100 ul (96-well plate) or 30 ul (384-well plate) TCW buffer per well. After the third wash briefly centrifuge the plate and remove all residual TCW buffer.

4. Add 6 ul of PCR-grade water and proceed with the standard DriverMap protocol for cDNA Synthesis and Amplification directly in TurboCapture plate. Synthesis of cDNA, second strand primer extension, and PCR with the DriverMap primer pools can all be run directly in the TurboCapture plate.