Automation of Stratagene Absolutely
RNA 96 Microprep Kit with the Bravo
Automated Liquid Handling Platform

Application NoteAuthors
Owen Hardy and Stephen Lappin
Agilent Technologies
Santa Clara, CA USAAbstract
A new method was developed to automate RNA isolation in a 96-well format
using the Bravo Automated Liquid Handling Platform and Absolutely RNA 96
Microprep Kit from Agilent Technologies (Automation Solutions and Stratagene
divisions, respectively). Current RNA isolation protocols require extensive
manual processing including repetitive pipetting and vacuum filtration steps.
Using Agilent’s VWorks Automation Control software, a script was written for
the Bravo Platform to aspirate, dispense, mix, and transfer sample from asource 96-well tissue culture plate to the binding and collection plates of theAbsolutely RNA 96 kit as well as to automate sample wash steps using anaccessory vacuum filtration unit on the Bravo Platform. RNA quality wasassessed on the Agilent BioAnalyzer 2100.

Technical Note

Introduction

Protocol Workflow

The Stratagene Absolutely RNA 96
Microprep Kit allows high throughput
isolation of total RNA from small
samples of cultured cells. This simple
method eliminates toxic phenol-chloroform extractions and time-consuming
ethanol precipitations by selectively
binding RNA from cell lysate on 96-wellsilica fiber matrix plates. However,there are repeated washes, filtration,and centrifugation steps that are
amenable to automation. The Bravo
Platform has nine plate decks or stations that can fully accommodate theAbsolutely RNA 96 protocol. Described
below are a set of three protocols, written
for the Bravo Platform to automaticallyhandle binding, DNase treating, washing,and isolated RNA elution.

The Absolutely RNA 96 protocol can bedivided into three major steps:

Materials

5. Vacuum filter• Agilent Bravo Automated Liquid
Handling Platform (G5409A)

DNase Treatment (optional)• 96 channel LT Disposable Tip Head(04730-202)• Agilent Vacuum Filtration Station(G5432A)• Agilent 96LT 200 µL sterile, filteredpipette tips (08585-102)• Agilent VWorks Automation
Control software• Stratagene Absolutely RNA 96
Microprep Kit (#400793 or #400794)• 96-well Tissue Culture dishes(e.g. Costar Flat Polystyrene, #3598)

A summary of the steps for each are
the following:

Using VWorks Automation Control software, three scripts were written foreach of the three major steps outlined.
Unique labware was first defined in thesoftware to allow the Bravo Platform toproperly position the tip head relative tothe deck platepad. Accessories such as
the vacuum filtration unit and tip boxes
are predefined in the software. All threescripts are run independently to accommodate the optional DNase treatment.

Lysis and Binding

Absolutely 96 Buffer Preparation

1. Aspirate media from tissue
culture dishReagents and buffers for the Absolutely
96 Microprep kit were prepared according to the user manual. Specifically, (1)lyophilized RNase-Free DNase I wasreconstituted with supplied buffer and a
100 µL aliquot diluted in 5 mL DNase
dilution buffer per 96-well plate, (2)wash buffers diluted to 1X concentration with 100% ethanol, (3) BME added
to lysis buffer, (4) 100% sulfolane diluted to 80% with RNase-free water.

1. Preparing the cell lysate and binding
to the silica matrix plate
2. DNase treating on column (optional)
3. Wash and Elution

2. Dispense lysis buffer to dish3. Dispense 80% sulfolane todish and mix
4. Transfer lysate to Absolutely 96
Binding plate

1. Dispense low salt wash toBinding plate
2. Vacuum filter
3. Centrifuge to dry filter
4. Dispense DNase to Binding
plate and incubate
Wash and Elution
1. Dispense high salt wash toBinding plate
2. Vacuum filter• Buffer reservoirs and/or 96-wellplates for reagent preparation andwaste disposal

3. Dispense low salt wash toBinding plate• Agilent Microplate Centrifuge or othertabletop plate centrifuge (G5405A)

5. Repeat 3 and 4• Sulfolane (Sigma # T22209)• Ethanol

4. Vacuum filter
6. Centrifuge
7. Dispense elution buffer toBinding plate and incubate
8. Centrifuge

2

For use with the Bravo Platform, it ispossible to use the following setup:
80% Sulfolane may be prepared inexcess for use in a buffer reservoir(20 mL is sufficient to process 2 runs in
96-well plate format but more isrequired when using a reservoir). For
DNase I, elution buffer, lysis buffer, andhigh salt buffer required the following
volumes per well: 50 µL, 30 µL, 100 µL,and 500 µL, respectively. This necessitates aliquotting to a 96-well plate fordispensing by the Bravo platform. TheBravo Platform can be used to prepare
these plates as well if desired. For low
salt wash, a reservoir filled with 200 mLfor each plate is sufficient.

Automation of Stratagene Absolutely RNA 96 Microprep Kit with the Bravo Automated Liquid Handling Platform

Centrifugation

Table 1: Bravo Setup Example

As required, plates were removed fromthe vacuum station on the Agilent Bravo
Platform and spun in a tabletop centrifuge at 1100 g. Alternatively, the plate
may be moved from the Bravo Platform
automatically and spun in the associatedAgilent Microplate Centrifuge.

Deck Position

RNA Analysis
Total RNA from 12 wells (4 corners andmiddle row) were run on the Agilent
BioAnalyzer 2100 using an Agilent RNA
6000 Nanochip, and assessed for sample
quality using BioAnalyzer 2100 Expert
software version B.02.06.

Binding

DNase Treat

1

Vacuum station
Vacuum station
with Absolutely 96 with Absolutely 96
Binding Plate
Binding Plate

2

Sample Tissue
Culture Dish3

Sulfolane reservoir4Wash/Elution
Vacuum station
with Absolutely 96
Binding Plate

DNase solution plate*

—

—

—

Low salt wash
reservoir

Low salt wash
reservoir

Low salt wash
reservoir

5Waste plate——

6Lysis buffer

7

—

—

High salt wash
reservoir

8

Filter Tip Box—

Filter Tip Box*

9

Filter Tip Box

Filter Tip Box*

Filter Tip Box*

Elution buffer** Plates need to be swapped out before the start of the protocol. Note that the Bravo Platform tip head may be equippedwith a plate gripper to move plates to a free platepad as required.

Figure 1. Absolutely RNA Kit Method

Figure 2. Bravo layout for lysis and binding steps

Figure 3. Bravo layout for DNase treatment step

Figure 4. Bravo layout for wash and elution steps

3

Results and Conclusion
After completion of the RNA isolation,
samples from the four corners andmiddle row of the 96-well tissue culture
dish were analyzed using the Agilent
BioAnalyzer 2100 to assess both thequality of the isolated RNA, as well as
the absence of genomic DNA contamination. See Figure 5 for representativeelectropherograms produced using
BioAnalyzer 2100 Expert software. In
this case, samples are overlaid, withthe four corners displayed in the topgraph, and the middle row in the bottomgraph. Note the RNA is fully intact withsharp peaks for 18S and 28S ribosomal
RNA, the presence of ‘small RNA’ peak,and lack of genomic DNA contamination.
RNA integrity numbers were also calculated by the 2100 Expert software andranged from 9.2 to 9.6.
In conclusion, these results demonstratethat the Agilent Bravo AutomatedLiquid Handling Platform can be usedsuccessfully to automate total RNA
isolation using the Absolutely RNA 96Microprep kit.

Figure 5: Electropherograms recorded using the Agilent BioAnalyzer 2100. Total RNA was isolated fromHeLa cell culture in a 96-well plate using the Agilent Bravo Automated Liquid Handling Platform andAbsolutely RNA 96 Microprep kit. Representative samples from the four corners (top) and middle row of
the plate (bottom) are overlaid. Samples were analyzed using Agilent 2100 Expert software and assessed
for absence of genomic DNA contamination and RNA quality. RNA integrity number (RIN) was determined by the software and ranged from 9.2-9.6.

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© Agilent Technologies, Inc. 2009
Published in the U.S.A. November 1, 2009
5990-3558EN