Complete Automation of Quantitative
Polymerase Chain Reaction Assays onthe Agilent Bravo Automated Liquid
Handling Platform
Application Note

Abstract
The Agilent Bravo Automated Liquid Handling Platform has been shown to be an effective
way to automate setup of a variety of quantitative polymerase chain reaction (QPCR) assays.
QPCR reactions to examine a variety of housekeeping genes from cDNAs derived from different
human tissues were set up on the Bravo Automated Liquid Handling Platform and by hand.
Dilutions performed by each method showed similar slopes and good linearity and targets
amplified by either method also gave similar Ct values. When examining cDNAs from fetal
and adult lung, differential expression was observed in the B2M whereas GUSB and TBP
remained similar. The Bravo Automated Liquid Handling Platform should be useful to scientists
interested in speeding up setup while maintaining reliability when performing QPCR studies.

IntroductionAuthors
David Knorr
Manuel Gomez
Stephen Lappin
Agilent Technologies, Inc.
Santa Clara, CA USAQPCR assays have become widespread for detecting and measuring the abundance of specificgenetic targets. Technologies developed to detect and monitor PCR product formation andaccumulation include TaqMan and Molecular Beacons, which measure accumulation of
PCR product-specific fluorescent probes. Another method involves measuring the increase offluorescent DNA intercalating dyes such as SYBR Green. Agilent Technologies provides tools
and reagents to speed reaction set-up while reducing possible mistakes and inaccuracies
that may arise from performing these steps manually. Here we describe using the Bravo
Automated Liquid Handling Platform to set up a series of SYBR Green QPCR experiments toexamine relative gene expression levels for a number of targets in several tissues.
The Bravo Automated Liquid Handling Platform has nine plate deck positions, which alongwith an Agilent BenchCel Workstation and an Agilent Microplate Centrifuge can fully automate
setup of the QPCR protocols without manual intervention. Described below is a protocol,
written for a workstation containing the Bravo, a BenchCel Microplate Handling Workstation
and an Agilent PlateLoc Thermal Microplate Sealer to automatically handle reagent dilution,aliquoting, and final assay plate preparation.

ExperimentalPrimers

First Strand cDNA mixtures derived frompoly[A] + total RNA from Human adult andfetal lung tissue, adult Heart and HeLa cells(each at 500 pg/µL) were used as templates.
A set of ten Human housekeeping geneprimers was obtained from RealTimePrimers(product number HHK-1). An additional setof primers specific for ß-Actin (includedwith Stratagene cDNAs) was also used.
Final reactions were prepared by combining
250 pg of cDNA template diluted to 10 µL inwater with a combination of one of thespecific primer pairs and master mix(Stratagene Brilliant II SYBR Green QPCR
Master Mix, Agilent Part Number 600828)
diluted to 15 µL. Each cDNA / target reaction
was prepared in duplicate. Identical reaction
mixes were prepared either by hand or using
the Agilent Bravo according to theStratagene recommendations. Reaction
plates were sealed automatically with aclear QPCR-compatible seal (Agilent Part
Number 17318-001) using an Agilent PlateLoc
Thermal Microplate Sealer. Reactions weredeveloped using a Stratagene Mx3005P
Real Time Thermal Cycler (G5105A) for
40 cycles according to the kit recommendations. Data was collected and processedusing MxPro v4.10 software.

Templates

To compare possible differences between
using a Bravo and hand-pipetting, cDNAfrom fetal lung tissue was diluted 1:2 seventimes to provide template concentrations
from 500 – 7.8 pg/µL. Linear regression
analysis of the Ct values plotted against
template starting concentration gave R2
values of 0.973 for the Bravo and 0.983 forthe hand-pipetted sets.

Add 15 µl
Primer Mix

Prepare Mixes

NTC

Prepare Reactions
Diluted Primers+Master Mix
Diluted Templates
Figure 1. General workflow used to prepare QPCR reactions.

Performance of Bravo- vs. Hand-Pipetted
2-fold Dilution Series
Bravo
Hand-pipetted

26

Log. (Hand-pipetted)
Log. (Bravo)

25
2423

Ct

Results and Discussion

Add 10µl Template (11)Master Mix

2221201918
110100pg total cDNA
Figure 2. Performance of Bravo vs. hand-pipetted 2-fold dilutions

21000

Differences between Agilent Bravo andhand-pipetting were also examined by looking
at a number of different housekeeping
genes. The plot shown below shows eachplot line as an average between replicate
wells for both the relative differences ofdifferent starting concentrations for different
gene targets.
The table below gives the absolute differences in Ct values between targets thatwere prepared by the Bravo and by hand.
Most targets gave very similar Ct values,indicating similar relative abundance foreach target.

Figure 3. Amplification plots for seven different housekeeping genes prepared using the Bravo,
or by hand in amplifying targets from fetal lung tissue. Target identities are given in the legend.

Comparison of Ct values obtained When Preparing Targets using the Bravo or by Hand
TargetFetal Lung

HeLa

Fetal Heart

Bravo Hand

D

Bravo Hand

D

Bravo Hand

D

ACTB

20.37

20.04

0.33

19.87

20.09

0.22

19.52

19.54

0.02

B2M

23.86

23.6

0.26

22.64

22.93

0.29

20.21

20.15

0.06

GAPDH

22.74

22.44

0.3

19.28

19.36

0.08

18.6

18.43

0.17

GUSB

26.46

26.64

0.18

25.12

25.18

0.06

25.32

25.27

0.05

HPRT1

29.77

29.91

0.14

25.86

26.33

0.47

25.92

25.790.13

PGK

26.47

26.35

0.12

22.39

22.61

0.22

22.77

22.62

0.15

PP1A

22.53

22.28

0.25

20.94

21.37

0.43

20.5

20.44

0.06

RPL13A

20.67

20.13

0.54

20.21

20.36

0.15

18.77

18.55

0.22

TBP

28.85

29.03

0.18

28.55

28.85

0.3

27.85

28.11

0.26

TFRC

29.51

29.71

0.2

24.66

24.91

0.25

27.28

27.4

0.12

ß-actin

20.78

20.83

0.05

20.53

20.84

0.31

17.52

17.75

0.23

3

The data show good correlation for eachtarget whether developed using the Agilent
Bravo or by hand.
Examples of conserved and differential gene
expression are given in Figure 4. While
genes for GUSB and TBP showed similar
profiles and Ct values (around 23 and 25.5
respectively), the serum protein beta-2
microglobin (B2M) shows an apparent
difference in expression level with theadult version showing an approximately
4-fold increase in expression over thefetal version.

Conclusion
The Bravo Automated Liquid Handling
Platform, along with an Agilent BenchCel
Workstation and Agilent Microplate
Centrifuge, can be used to quickly automate
QPCR assays without sacrificing data qualitywhile greatly increasing overall throughput.

Figure 4. Amplification of three genes in fetal and adult lung. Ct values for each target are given in the legend.

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For Research Use Only. Not for use in diagnostic
procedures. Information is subject to change
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Agilent Technologies shall not be liable for errors
contained herein or for incidental or consequential
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© Agilent Technologies, Inc. 2009
Published in the U.S.A. August 21, 2009
5990-4522EN