MagNA Pure LC: Evaluation as a Sample Preparation System for … · MagNA Pure LC: Evaluation as a Sample Preparation System for the LightCycler Instrument 10-16 MagNA1zi - 3ak.fm

BIOCHEMICA · No. 1 n 200010 ROCHE MOLECULAR BIOCHEMICALS

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Laurence Lapopin and Michael Kirchgesser

Roche Molecular Biochemicals, Penzberg, Germany

The MagNA Pure LC is an instrument for fully auto-mated purification of nucleic acids (DNA, RNA, andmRNA) from different sample types and volumes. Thepurification principle is based on the selective nucleicacid adsorption onto magnetic glass particles (MGPs),producing high quality nucleic acids. The instrumenthas walk-away processing and can perform 32 nucleicacid purifications in less than 60 minutes. The MagNAPure LC requires no hands-on time after starting theprotocol, and even the pipetting of subsequent PCRscan be done automatically. Therefore, the risk of con-tamination is significantly reduced which is crucial forall PCR analyses, and the reproducibility of the resultsis improved. Moreover, due to the flexibility of the soft-ware, sample and elution volumes can be easily varied.The high quality of isolated nucleic acids allows youto perform numerous downstream applications suchas PCR and RT-PCR analysis, enzymatic digestion, aswell as Southern and Northern blot analysis.

Since its introduction in 1998, the LightCycler Systemhas been synonymous with extremely fast, accurate,and reliable PCR analysis. Up to 32 samples can beamplified by on-line real-time PCR, and the productscan be directly quantified [1].

The MagNA Pure LC is a robotic workstation for nucleicacid isolation, designed to efficiently complement theLightCycler System. The MagNA Pure LC combinesrapid nucleic acid purification from up to 32 sampleswith direct filling of the LightCycler Capillaries or 96-well PCR plates, and is suitable for the most common-ly used PCR instruments. The fully automated MagNAPure LC includes easy-to-use software that controlsnot only all instrument functions and nucleic acid isola-tion steps, but also permits the transfer of the sampleinformation and data from the workstation to theLightCycler Instrument. That makes the MagNA PureLC the perfect upstream instrument for the Light-Cycler System.

The automated processing of the MagNA Pure LC re-sults in a walk-away system, performing 32 nucleic acidisolations in a minimum amount of time (i.e., less than60 minutes). The MagNA Pure LC nucleic acid purifi-cation is based on magnetic glass bead technology,eliminating centrifugation and vacuum steps. Thisfeature combined with the automated processing sig-nificantly reduces the cross contamination risk. Al-though the MagNA Pure LC is a fully automated anda highly controlled instrument, it leaves considerableflexibility to the user. The system allows to performnucleic acid isolations from various starting materials(e.g., blood, white blood cells, cultured cells, andtissue section) with a high variability in sample andelution volumes. Moreover, the procedure can auto-matically detect clots and tip-loss, allowing a walk-away during the purifications.

In this article, we describe the first results obtainedwith the prototype version of the MagNA Pure LC.Genomic DNA and total RNA were isolated from dif-ferent sample types and volumes (20 to 200 µl bloodand 102 to 106 culture cells). The instrument perform-ance was checked by evaluating its reproducibility, theintegrity and the purity of the isolated nucleic acids, aswell as potential cross contamination. The high qual-ity of the purified nucleic acids was checked by PCRand RT-PCR analysis, restriction enzyme digestion,and Northern blot analysis.

Description of the instrumentThe inside part of the workstation is divided into 3 distinctcompartments. As shown in Figure 1, the left compart-ment contains the reagent tubs, the 4 x 8-well samplecartridge, and the pipette tips. The samples are pro-cessed in the central compartment. Finally, the rightcompartment contains 2 temperature-regulated blocks;the elution (70°C) and storage block (7°C). This com-partment also contains the post elution block which ispermanently cooled to 7°C, here the PCR set-up is done,either in the LightCycler Capillaries in the carousel orin the centrifuge adapters, or in 96-well PCR plates,strips, or single tubes.

AAbstract

IIntroduction

MMaterial and Methods

MagNA Pure LC: Evaluation as a Sample Preparation System for the LightCycler Instrument

10-16 MagNA1zi - 3ak.fm Seite 10 Freitag, 14. Januar 2000 2:12 14

ROCHE MOLECULAR BIOCHEMICALS BIOCHEMICA · No. 1 n 2000 11

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The MagNA Pure LC instrument is associated with re-agent kits that contain all the reagents required forthe nucleic acid isolation from blood and cells.

Nuclease-free containers are filled with the appropri-ated volume of reagents and placed in a specific po-sition in the workstation, as described by the supplier.The sample cartridge (4 x 8 wells) is filled with thesamples and placed into the workstation. After theinstrument is set-up, no more hands-on steps are re-quired. The robot dispenses all reagents in the proces-sing cartridges. Purification and isolation proceduresare then carried out automatically following a specificprogram previously selected in the software.

Genomic DNA isolation from blood and cellsThe MagNA Pure LC isolates genomic DNA from 102

to 106cells suspended in 200 µl PBS, as well as from20 to 200 µl of human blood.

The samples are incubated in a special buffer (Lysis/Binding Buffer) which inactivates nucleases and thecell lysis is performed by pipetting up and down. Pro-teinase K is added to the lysate to digest the cellularproteins. Next, magnetic glass particles (MGPs) aremixed with the lysate. The released DNA adsorb ontothe bead surface due to the chaotropic salt conditionsand the high ionic strength of the Lysis/Binding buffer.Cellular debris and unbound substances which ofteninhibit PCR reactions (proteins, nucleases, heparin,and hemoglobin), are removed with Washing Buffer I.Washing Buffer II is used to remove any remainingimpurities and to reduce the chaotropic salt concen-tration. The DNA is finally eluted at 70°C in a low-saltbuffer with a flexible elution volume range from 25 to200 µl. The DNA is then transferred to a cooled storagecartridge (Figure 2).

Total RNA isolation from blood and cellsThe MagNA Pure LC isolates total RNA from 102 to106 cells suspended in 200 µl Lysis/Binding Buffer.The instrument also isolates RNA from white bloodcells (WBCs) harvested from 20 to 200 µl human bloodand suspended in Lysis/Binding Buffer.

The samples are incubated in the Lysis/Binding Bufferto inactivate the nucleases and lyse the cells. The ma-gnetic glass particles (MGPs) are added to the lysate,

and the released DNA and RNA adsorbonto the bead surface. The nucleic acid-coated beads are then transferred to a low-salt DNase solution and DNA is digested.The whole mix is added to fresh Lysis/Bind-ing buffer to rebind the RNA moleculesonto the beads surface. Then Washing Buf-fers I and II are added successively to re-

› Figure 1: The MagNA Pure LC workstation.

¤ Figure 2: Principle of genomic DNA isolation. Genomic DNA isola-tion from various biological materials is achieved in 5 steps: 1: sample setup; 2: cell

lysis; 3: nucleic acid adsorption onto bead surface; 4 + 5: several washes and ma-

gnetic bead purification; 6: nucleic acid elution in low-salt buffer



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move any cellular debris and potential inhibitors, asdescribed in the previous paragraph.

Total RNA is eluted at 70°C in a low-salt buffer in aflexible volume ranging from 25 to 200 µl. The RNA isstored at 7°C on the cooling block (Figure 3).

Post-elutionAfter nucleic acid isolation, the set-up of the PCR andRT-PCR reactions is performed by the MagNA Pure LC.The isolated nucleic acids and PCR/RT-PCR mastermixes can be automatically pipetted into LightCyclercapillaries, strips, tubes, or 96-well PCR plates.

Other Downstream applicationsDNA was digested by commonly used restriction en-zymes according to the enzyme supplier protocol. Iso-lated DNA and RNA was also used for typical South-ern and Northern blot analysis, respectively, asdescribed by Sambrook et al [2] and Farrell [3].

Analysis of nucleic acid qualityThe performance of the MagNA Pure LC was firsttested by evaluating the quality and the purity of theisolated nucleic acids. DNA was prepared from cellsand from human blood. The gel electrophoresisrevealed that genomic DNA from cells (Figure 4A) aswell as from blood (Figure 4B) is of high quality with

a molecular weight greater than 20 kb. Moreover, thegel picture of the cellular DNA samples showed thatRNA is co-eluted with DNA.

Total RNA was isolated from culture cells and fromWBCs harvested from human blood. The gel electro-phoresis of RNA isolated from culture cells (Figure 4C)revealed the typical 2 bands corresponding to the 28Sand 18S rRNA and suggested the absence of DNAcontamination. The gel also clearly showed that thefirst band was twice as intense as the second, indi-cating high RNA integrity.

The purity of the nucleic acids was estimated by theOD 260/280 nm ratio. For DNA isolated from the dif-ferent sample types (cells and blood), the ratio was1.8 ± 0.2 (Table 1A). High ratio values (close to 2.0)could be explained by the presence of co-eluted RNA,as shown in Figure 4A. The OD 260/280 ratio calcu-lated for total RNA isolated from cells was 1.9 ± 0.1,confirming the high quality of the isolated material(Table 1B).

Yields of DNA from culture cells and human bloodand yields of total RNA from culture cells were deter-mined by OD measurement at 260 nm (Tables 1A and1B). The amount of total RNA from WBCs harvestedfrom human blood was not big enough to be detect-able by the spectrometer. Therefore, yields of mRNAof the corresponding material were estimated byquantitative RT-PCR (Table 1B). Moreover, the datashowed that yields of DNA and RNA prepared fromdifferent amounts of sample material mirrored theamounts of the starting material.

These results demonstrated that the MagNA Pure LCisolates high quality DNA and RNA with high yieldsand purity, also preserving the scalability of the start-ing amount of material. The magnetic bead technologyenables the instrument to achieve such perfor-mances.

Intra-assay variance analysisThe intra-assay variance was determined by purifyingDNA from 32 samples of 20 µl human blood with theMagNA Pure LC in one single run (Figure 5). Thecoefficient of variance (CV%) was calculated for theyield (Figure 5A) and for the LightCycler Instrumentcrossing point (Figure 5D). For both, the CV% valueswere lower than 10%, indicating high reproducibilityof the nucleic acid isolation by MagNA Pure LC. Thisclearly demonstrates the reliability of the MagNa PureLC workstation when performing 32 nucleic acidisolations.

RResults and Discussion

› Figure 3: Principle of total RNA isolation. Total RNA isolation from various biologicalmaterials is achieved in 7 steps: 1: sample setup, 2: cell lysis, 3: nucleic acid adsorbtion ontobead surface, 4 to 6: DNase treatment and RNA rebinding to the beads surface, several washingsteps, 6: nucleic acid elution in low-salt buffer, 7: several washing steps, 8: nucleic acid elutionin low-salt buffer



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› Figure 4: Analysis of DNA and RNA isolated from cells and human blood.Gel electrophoresis of DNA and RNA isolated from cells and blood. 15 µl genomic DNA isolated from cells (A) and from blood (B), and 40 µl

total RNA (C) isolated from cells, were separated by 1% TAE/agarose gel electrophoresis. A molecular weight marker (MW) was run in

parallel.

A: DNA from K 562 cells

B: DNA from whole blood

C: Total RNA from K 562 cells

28 S

18 S

21.2 kbp

DNA

18 S

28 S

Number ofK 562 cells

DNA yield[mg]

Volume ofhuman blood

DNA yield[mg]

106 5.98 ± 1.5 20 µl 1.01 ± 0.17

105 1.30 ± 0.36 50 µl 2.32 ± 0.23

104 0.47 ± 0.03 100 µl 3.50 ± 0.20

103 200 µl 5.50 ± 0.35

102

➝ Ratio (OD 260/280) = 1.8 ± 0.2

Number ofK 562 cells

RNA yield[mg]

WBCs [xml]from

human blood

mRNA amount[ng] determined by

LightCycler

106 4.97 ± 0.40 25 µl 10.60 ± 0.050

105 1.90 ± 0.07 50 µl 16.16 ± 0.130

100 µl 19.66 ± 0.002

200 µl 28.12 ± 0.182

➝ Ratio (OD 260/280) = 1.9 ± 0.1

› Table 1A: Table of DNA yields. Genomic DNA was isolated from 102 to106 culture cells and from 20 to 200 µl human blood and recovered in 100 µl elu-

tion buffer. The samples were diluted 1:2 in the same buffer and submitted to OD

measurement at 260 and 280 nm. The amount of DNA was calculated for 100 µl

elution volume and corresponds to the average value calculated from 2 and 4 re-

petitions for cell and blood samples, respectively. The amount of DNA from 10 2

and 103 cells was not high enough to be detectable by the spectrophotometer.

› Table 1B: Table of RNA yields. Total RNA was isolated from 105 to 106culture cells and from white blood cells (WBCs) harvested from 25 to 200 µl hu-

man blood, and recovered in 100 µl elution buffer. The RNA from cells was dilu-

ted 1:3 and submitted to OD measurement at 260 and 280 nm. WBCs do not

contain enough RNA to be detectable by the spectrophotometer. Therefore the

yields of mRNA was estimated by quantitative RT-PCR in the LightCycler by am-

plifying the cyclophilin A transcript.

Cross-contamination analysisA cross-contamination experiment was carried out where16 samples with 200 µl human blood and 16 sampleswith 200 µl phosphate buffered saline (PBS) were usedas starting material for DNA isolation. In order tosimulate the conditions where cross-contamination wasmost likely, each blood sample in the sample cartridgewas surrounded by a well filled with PBS (Figure 6).

After DNA isolation, the 32 samples were used to ampli-fy the cyclophilin A gene in the LightCycler Instrument(Figure 6A). PCR products were also checked by gelelectrophoresis (Figure 6B). The results clearly show-ed that no product was present in the PBS samples,indicating that the 32 DNA isolations were performedwithout cross-contamination. The absence of cross-contamination is achieved by the use of a drop catcher



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placed under the 8-nozzle head, which avoids thespilling of samples during the isolation process. Addi-tionally, the inside of the instrument can be easily cle-aned with commonly used disinfectants and deconta-minated with a built-in UV-lamp inside the instrument.

Scalability analysisGenomic DNA isolated from different numbers ofcells were used to amplify the cyclophilin A gene withthe LightCycler Instrument. The results of the PCR arepresented in Figure 7. The PCR curves showed a reg-ular shift, and the crossing point values showed, a li-near increase. Both results mirrored the difference inthe starting material amount.

Similarly, total RNA isolated from WBCs harvested fromdifferent human blood volumes was used to amplifythe cyclophilin transcript in a one-step RT-PCR withthe LightCycler Instrument (Figure 8, A1). Additional-ly, in order to check the presence of DNA contamina-tion, a minus-RT control-PCR was done where the re-verse transcriptase was not added in the PCR mastermix (Figure 8, A2). The results of the minus-RT con-trol-PCR, confirmed the absence of DNA since theLightCycler graphs showed flat lines. The crossingpoint value analysis of the RT-PCR showed a linearincrease, also reflecting in this case, the differentstarting material amounts.

B

A

C

D

› Figure 5: Reproducibility analysis of the DNA isolated from 20 ml blood. DNA from32 x 20 µµ l human blood was isolated with the MagNA Pure LC in one single run. The DNA yieldcoefficient of variance (CV%) was calculated after OD measurement of the 1:2 diluted sample

eluates at 260 nm (A). The genomic DNA was then separated by 1% TAE/agarose gel electro-

phoresis (B). A Molecular Weight Marker (MW) was run in parallel. 5 µl DNA eluate was used

to amplify the cyclophilin A gene by the LightCycler with the Hybridization Probes format (C).

The crossing point coefficient of variance (CV%) was deduced (D).

A

B

› Figure 6: Cross-contamination analysis of DNA isolated from 200 ml blood. Sixteen wells of the sample cartridge were filled with200 µl blood (blocks 1 and 3, positions a, c, e and g, and blocks 2 and 4, positions b, d, e, and h) and neighbouring wells with 200 µl

phosphate buffered saline (PBS). Thus, 32 nucleic acid isolations were carried out in one single run by the MagNA Pure LC, simulating

the conditions under which cross-contamination was most likely to occur. Five µl eluate was used for amplification by LightCycler PCR

using Hybridization Probes and cyclophilin A as target gene (A). The PCR products were separated by 1% TAE/agarose gel electrophoresis

(B). A molecular weight marker (MW) was run in parallel.



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These results demonstrated that the MagNA Pure LCisolates genomic DNA and total RNA, strictly conservingthe scalability of the starting material even after PCRor RT-PCR. This is a fundamental requirement, i.e. forresearchers studying gene expression based onquantitative RT-PCR.

Restriction enzyme digestion and blotting analysis1.5 µg DNA isolated from 200 µl human blood was di-gested by the restriction enzyme Eco RI, in order tocheck the digestibility of the DNA purified with the in-strument. The results are presented in Figure 9. Thedigested samples showed a clear smear compared tothe non-digested DNA, indicating that DNA can beefficiently digested, a prerequisite, for example forSouthern blotting.

Similarly, a β-actin digoxigenin-labeled probe was usedin Northern blot analysis for evaluating the quality ofthe RNA isolated from cells (Figure 10). The picture ofthe hybridization results showed for each sample onedistinct band of the expected size (1.8 kb). This resultdemonstrates that RNA isolated with the MagNA PureLC is not degraded and of high integrity.

Figure 8: RT-PCR analysis of RNA isolated from WBCs.5 µl RNA eluate from WBCs harvested from blood was used for ampli-

fication of the cyclophilin A gene by LightCycler PCR using the Hy-

bridization Probe format (A1). A minus-RT control PCR was performed

in order to show the absence of DNA contamination. Standards of 50 ng,

10 ng, and 5 ng DNA were amplified in parallel (A2). The crossing

point (CP) average values were calculated from 3 repetitions (B) and a

graphic was deduced (C).

C

A1

A2

B

A

B

C

› Figure 7: PCR analysis of cellular DNA. 5 µl DNA eluate from 106 to 102 cells (see Figure4) were used for amplification of the ββ-globin gene by LightCycler PCR using HybridizationProbe format (A). The crossing point (CP) average values were calculated from 4 repetitions (B)

and a graphic was deduced (C).



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These experiments demonstrated that the MagNAPure LC prepared high quality DNA and RNA allowingyou to reliably perform the most common down-stream applications.

The MagNA Pure LC is an extremely fast and accurateinstrument, able to perform up to 32 high quality nucleicacid isolations and subsequent PCR set-up. The assess-ment of the MagNA Pure LC performance was describedin this article. Nucleic acids were isolated from dif-ferent sample types (20 to 200 µl human blood and102 to 106 culture cells). The nucleic acid analysis con-firmed the high quality of the isolated DNA and theRNA. It was demonstrated that the isolation processbased on the magnetic bead technology preserves thescalability of the starting material amount. This latter

performance is a crucial requirement for all analysesof gene expression, either by quantitative RT-PCR orby Northern blot. The purified nucleic acids wereshown to be free of cross-contamination, allowing areliable downstream analysis based on PCR. The iso-lated RNA is free of contaminating DNA due to the ef-ficient DNase digestion. Moreover, the experimentsclearly demonstrated the ability of the MagNA PureLC to isolate nucleic acids with high reproducibilitywhich is a fundamental need for the field of medicalresearch. The MagNA Pure LC is also an instrumentwith high flexibility. It allows to purify DNA, total RNA,as well as mRNA from a broad variety of samples suchas blood, WBCs, peripheral blood mononuclear cells,or culture cells. Different sample volumes (20 to 300 µl)and cell numbers (102 to 106) can be used as startingmaterial, and the nucleic acids can be recovered invariable elution volumes (25 to 200 µl). The high qual-ity of the isolated nucleic acids allows to performnumerous downstream applications. Therefore, theperformance of the MagNA Pure LC in isolating nucleicacids makes this workstation not only the perfectupstream instrument for the LightCycler System, butalso a reliable and accurate upstream instrument forall research laboratories working on nucleic acidanalysis.

References[1] Wittwer, C.T., Ririe, R.V., David, D.A., Gundry, R.A., and

Balis, U.J. (1997) BioTechniques 22: 176–181

[2] Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual. Cold Spring Harbor Labora-

tory Press. Cold Spring Harbor, NY.

[3] Farrell, R.E. (1993) RNA Methodologies: A Laboratory Guide

for Isolation and Characterization, Academic Press, San Diego.

CConclusion

› Figure 10: Northern blot analysis of the b-actin ex-pression on RNA isolated from cell. Two µg total RNA isola-ted from 106 cells (1 and 2) was separated by denaturing agarose gel

electrophoresis (A), transferred onto a nylon membrane and hybri-

dised with the ß-actin DIG-labelled probe (B).

¤ Figure 9: Digestibility of genomic DNA isolated fromblood. 1.5 µg DNA eluate was digested by Eco RI at 37°C for 4 h (Aand B). The control without enzyme is shown in C. The Eco RI di-

gested samples, non-digested samples, and a molecular weight

marker (MW) were separated by 1% TBE/agarose gel electro-

phoresis.


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MagNA Pure LC: Evaluation as a Sample Preparation System for … · MagNA Pure LC: Evaluation as a Sample Preparation System for the LightCycler Instrument 10-16 MagNA1zi - 3ak.fm