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Automated Total RNA Purification From Human Lung Carcinoma Cells on the Biomek® 3000 and Biomek® NX Laboratory Automation Workstations

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Ruth Zhang Ph.D., Chad Pittman, Laura Pajak Ph.D., and Graham Threadgill Ph.D.
Beckman Coulter, Inc., Indianapolis, Indiana and Fullerton, California

Purpose:

To automate total RNA purification process from human lung carcinoma cells (A549).

Methods:

Methods for Promega's SV96 Total RNA Isolation System were developed for the Biomek 3000 and Biomek NX, the new, automated liquid handlers from Beckman Coulter, Inc. Both automated total RNA purification protocols can generate high quality and high purity total RNA in the 96-well plate format within 30-70min without user intervention. DNase treatment is included in the protocol so that the resulting total RNA can be used directly for down-stream applications without further manipulation, such as RT-PCR*.

Click image to enlarge
Figure 1. Biomek 3000 Protocol for RNA Purification.
Figure 1. Biomek 3000 Protocol for RNA Purification.

Biomek System	Pod	Wash Tool	Partial Plates?	Throughput	Method Time
Biomek 3000	MP 200	Yes	Yes	Low	70 min.
	Gripper Tool
Biomek NX	Multichannel	No	No	Medium	30 min.

Table 1. Comparison of total RNA Purification methods on the Biomek 3000 and the Biomek NX.

Figure 2. Starting instrument setup for total RNA purification on the Biomek 3000.

Figure 3. Starting instrument setup for total RNA purification on the Biomek NX.

Figure 4. Agarose gel electrophoresis of total RNA purified from Biomek 3000. Lane 1: MW Marker; Lane 2-12: total RNA of A549 cells.

Workstation	Cells/Well	A_{260/280 Ratio}/3 Plates	Average Yeld (ng/well)
Biomek 3000	50,000 - 60,000	1.99 +/- 0.0073	1044 +/- 198.48
Biomek NX	50,000 - 60,000	1.94 +/- 0.0102	1248 +/- 276.25

Table 2: Quantity and quality of RNA from A549 cells using the SV96 Total RNA Isolation System (Promega, Inc.) on the Biomek NX and Biomek 3000.

Results:

A549 cells, a human lung carcinoma cell line, were grown in 96-well plates for purification. The average concentration of purified total RNA from Biomek 3000 and Biomek NX liquid handlers was approximately 13-15 ng/µL from 0.5 x 106 cells/well with an average yield of approximately 1 µg/well using the default elution volume of 100 µL.