Applications

In this application note the integration of the PureLink RNA Purification Kit into the epMotion 5075 automated pipetting system is described. The PureLink 96 RNA Purification System provides high-throughput isolation of high-quality total RNA from the widest range of sample types and sizes. It is possible to isolate high yields of total RNA with low genomic DNA contamination from cultured cells, bacteria, yeast, plant, or mammalian samples. Cultured 293F cells (5 x 105 cells/well) were used as starting material. Typical yield was up to 25 µg per well. The total processing time on the epMotion 5075 VAC for 96 samples was 48-53 min.

Note 179 | December 2010

Eric C. Olivares, Invitrogen Corporation, Carlsbad, CA 92008, USADaniel Wehrhahn, Eppendorf AG, Hamburg, Germany

Automated High-Throughput Purification of total RNA using the Purelink™ 96 RNA Purification Kit on the Eppendorf epMotion® 5075 VAC automated pipetting system

Introduction

Abstract

Automated processing of nucleic acid purification can dra-matically increase laboratory throughput while reducing sample-to-sample variation and thereby increasing the reli-ability of the results. Invitrogen’s Purelink silica filter plate kits in combination with the Eppendorf epMotion 5075 VAC automated pipetting system offer a complete solution for processing 96-well filter plates in a vacuum format. This cost effective solution can be installed within a few hours in the laboratory.

Recently, protocols for genomic DNA purification from blood samples and mouse tails, PCR clean up and total RNA puri-fication have been optimized for use on the epMotion 5075 system. Here we present application data for the

PureLink 96 RNA Purification Kit. The PureLink 96 RNA Purification System provides high-throughput isolation of high-quality total RNA from the widest range of sample types and sizes. It is possible to isolate high yields of total RNA with low genomic DNA contamination from bacte-ria, yeast, plant, or mammalian samples. RNA binds to a silica membrane and impurities are removed by washing through vacuum filtration or centrifugation. High-purity total RNA is eluted into RNase-free water and ideal for use in a variety of applications, including RT-PCR, qPCR, northern blotting, and nuclease protection assays. The PureLink 96 RNA Purification System is supplied as a complete kit that includes filter plates, receiver plates, lysis buffer, Wash Buffers I and II, and RNase-free water.

Application Note 179 | page 2

Materials and Methods

Cultured 293F cells (5 x 105 cells/well) were lysed manu-ally and the lysate was transferred to a deep-well plate for automated processing. It is possible to perform the lysis step directly on the instrument, however due to the labile nature of RNA it is preferable to lyse the cells immediately after harvesting to stabilize the cellular transcriptome. A representation of the worktable set-up is shown in Figure 1. Tables 1 and 2 show the required disposables, epMotion accessories and labware. Table 1 also indicates the Buffer positions within the reagent rack. Table 3 shows a descripti-on of the automated method commands.

Figure 1: epMotion 5075 VAC worktable setup for the PureLink 96 RNA Purification Kit

Table 1: epMotion 5075 VAC worktable details for the PureLink 96 RNA protocol

Table 2: Required epMotion 5075 VAC accessories for the Pure-Link 96 RNA protocol

Table 3: epMotion programm description for the total RNA purification protocol

Position Labware Comment

T0 Gripper Tool for moving

T1 TM 1000-8 1000 µL 8-channel pipetting tool

A2 ep T.I.P.S. Motion 1000 µL 96 tip rack

A3 ep T.I.P.S. Motion 1000 µL 96 tip rack

A4 Eppendorf Deepwell Plate 96/1000

B2 ep T.I.P.S. Motion 1000 µL 96 tip rack

B3 Reagent Reservoir Module

Position 1: Lysis Buffer 30 mL reservoir

Position 2: 70 % Ethanol 30 mL reservoir

Position 3: Wash buffer 1 100 mL reservoir

Position 4: Wash buffer 2 100 mL reservoir

Position 5: Wash buffer 2 100 mL reservoir

Position 6: Elution Buffer 30 mL reservoir

VACUUM On frame: Purelink 96 RNA Purification Plate Included with Invitrogen kit

Vacuum Frame 1 Top collar for vacuum chamber

Inside manifold: 400 mL reservoir with Channeling Plate Waste container

TEMP3 Eppendorf Deepwell Plate 96/1000 Input samples

Height Adapter 55 mm (optional)

C4 Vac Frame Holder

Step Command Purpose

1 Number of samples Setup input sample number

2 Reagent transfer Dispense Lysis Buffer (150 µL)

3 Reagent transfer Dispense ethanol

4 Sample transfer Mix samples and transfer to filter plate

5 Vacuum (900 mbar) Bind samples (2 min)

6 Reagent transfer Dispense Wash Buffer 1 (950 µL)

7 Vacuum (900 mbar) Bind samples (2 min)

8 Reagent transfer Dispense Wash Buffer 2 (950 µL)

9 Wait 1 min

10 Vacuum (900 mbar) Bind samples (2 min)

11 Reagent transfer Dispense Wash Buffer 2 (950 µL)

12 Vacuum (900 mbar) Bind samples (2 min)

13 User intervention Tap plate on paper towels (optional)

14 Vacuum (900 mbar) Drying step (10 min)

15 Transport Remove vacuum lid and filter plate

16 Transport Remove waste container from vacuum manifold

17 Transport Replace elution plate in vacuum manifold

18 Transport Replace vacuum lid and filter plate

19 Reagent transfer Dispense elution buffer (170 µL)

20 Wait 1 min incubation for elution

21 Vacuum (900 mbar) Elution

22 End of protocol

Accessories required Quantity

Hardware Gripper

1000 µL 8-channel pipetting tool

Reagent Reservoir Module

Vacuum Frame 1

Vac Frame Holder

400 ml reservoir with Channeling Plate

Height Adapter 55 mm (optional)

Consumables ep T.I.P.S. Motion Filter 1000 µL 1.4 boxes/plate

30 mL reagent reservoirs 3/run

100 mL reagent reservoirs 3/run

Eppendorf Deepwell Plate 96/1000 1-2/run

Application Note 179 | page 3

Figure 2: 5 × 105 293F cells per well were processed using the PureLink 96 TotalRNA Purification Kit on the epMotion 5075 VAC. Yield was quantified using the fluorescence-based Quant-iT RNA Assay Kit. Observed %CV across the plate (n = 80) was 12.5 %, with a mean yield of 10.1 ± 1.2 µg.

Results

Samples were eluted in 170 µL into an Eppendorf Deepwell Plate 96/1000. Total processing time was approximately 45 minutes from start to finish. Yield was evaluated using aga-rose gel electrophoresis and the Quant-iT dsDNA Assay Kit (Broad Range). Yields were measured using the Quant-iT RNA Assay Kit and are depicted in Figure 2.

Figure 3: Agarose gel electrophoresis of total RNA isolated with the PureLink TotalRNA Kit from 5 × 105 293F cells. Total RNA (15 µL of eluate, ~9% of total yield) was separated on a 1 % agarose EGel.

Figure 4: Agilent Bioanalyzer analysis of total RNA integrity. RNA prepared from 5 x 105 293F cells using the PureLink 96 To-tal RNA Kit (1 µL, ~50 ng) was subjected to microcapillary elec-trophoresis using the Agilent Bioanalyzer RNA Nano 6000 chip system. Control RNA samples (~250 ng) were also included. The Bioanalyzer-calculated RNA Integrity Numbers (“RIN”) are from measurements of the electrophoretic trace. The PureLink samples shown all resulted in RINs of 10, indicating the highest possible integrity. Lane 1: WHICH Ladder, Lanes 2-3: control RNA, 250 ug; Lanes 4-7: PureLink RNA samples (D1-D4).

Figure 5: First-strand cDNA was synthesized from ~ 300 ng of total RNA using reagents included in the Invitrogen Super-Script III First-Strand Synthesis SuperMix for qRT-PCR kit. A ten-fold dilution series was prepared (1:101 through 1:106), and 5 µL of each dilution was used in triplicate qPCR reac-tions for beta-actin. The certified LUX primer set for beta-actin (Invitrogen) was used in combination with Platinum Quantitative PCR SuperMix-UDG with ROX (Invitrogen). Real-time detection of amplification was performed using an ABI 7900HT thermo-cycler. A. Standard curves were plotted for four random samples over the 6-log dilution series, and all four samples show a squared correlation coefficient (r²) of between 0.997–0.999, demonstra-ting robust performance of the isolated RNA in qRT-PCR. B. The amplification plot for sample C5 is shown below.

n = 80Mean Yield (ug) = 10.1 ± 1.2%CV = 12.5%

0 10 20 30 40 50 60 70 80

Well Position

20181614121086420

Yie

ld (

ug

)

Resulting total RNA samples were subjected to a variety of quality measurements. Agarose gel electrophoresis (Figure 3) and Agilent Bioanalyzer analysis (Figure 4) demonstrate that the isolated RNA is of high integrity and contains mini-mal genomic DNA contamination. Quantitative RT-PCR for a housekeeping gene was used to assess the performance of the isolated total RNA in downstream applications. As shown in Figure 5, the RNA produces linear amplification plots in qRT-PCR down to a cDNA dilution of 1 × 106-fold.

45

40

35

30

25

20

Ct

log ([cDNA])

0 1 2 3 4 5 6 7

y = -3.6846x + 44.813

R2 = 0.999

log ([cDNA])

0 1 2 3 4 5 6 7

45

40

35

30

25

20

Ct

y = -3.531x + 43.917

R2 = 0.9991

log ([cDNA])

0 1 2 3 4 5 6 7

45

40

35

30

25

20

Ct

y = -3.5841x + 43.825

R2 = 0.9988

log ([cDNA])

0 1 2 3 4 5 6 7

45

40

35

30

25

20

Ct

y = -3.6905x + 44.327

R2 = 0.997

Amplification Plot

0 5 10 15 20 25 30 35 40 45 50Cycle

1.000

1.000 E-1

1.000 E-2

1.000 E-3

∆ R

n

Your local distributor: www.eppendorf.com/worldwideEppendorf AG · 22331 Hamburg · Germany · Tel: +49 40 53801-0 · Fax: +49 40 538 01-556 · E-mail: eppendorf@eppendorf.com

Eppendorf North America, Inc. · 102 Motor Parkway · Hauppauge, N.Y. 11788-5178 · USATel: +1 516 334 7500 · Toll free phone: +1 800-645-3050 · Fax: +1 516 334 7506 · E-mail: info@eppendorf.com

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Application Note 179

Ordering Information Eppendorf

Ordering Information InvitrogenProduct Cat.#.PureLink™ 96 RNA Purification Kit, 4 x 96 preps 12173-011

Product NoteOrder no. international

Order no.North America

epMotion® 5075 VAC 230 V with integrated vacuum station 5075 000.016 N/AepMotion® 5075 VAC 120 V with integrated vacuum station N/A 960020014Thermoblock for PCR plates 96 well semi-skirted 5075 766.000 960002083Channeling Plate 5075 794.004 960002540Dispensing tool TM 1000-8 5280 000.258 960001061Reservoir Rack 5075 754.002 960002148Reservoirs 100 mL 10 x 5 reservoirs in bags/case, PCR clean 0030 126.513 960051017Reservoirs 30 mL 10 x 5 reservoirs in bags/case, PCR clean 0030 126.505 960051009Height Adapter 55 mm 5075 752.000 960002113Deepwell Plate 96/1000 DNA LoBind 20 plates 0030 503.201 951032808

Conclusion

The PureLink RNA Purification Kit can be successfully auto-mated using the easy-to-use Eppendorf epMotion 5075 VAC automation system. The purified RNA is of excellent integrity and is well suited for various downstream applications inclu-ding Quantitative RT-PCR. Invitrogen developed and tested this protocol together with Eppendorf and will

provide full application support for any questions. The method and labware file required to run this method can be downloaded from the Plug´n´Prep website www.eppendorf.com/pnp. The files can be transferred to the epMotion software within minutes making this method a ready to go solution.

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