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Total RNA Purification from Cultured Cells Using the ABI PRISM 6700 Automated Nucleic Acid Workstation and Total RNA Lysis Reagents

Abstract

Applied Biosystems has developed new techniques that produce highpurity total RNA from biological sources such as cultured cells. These techniques eliminate the need for post-isolation DNase treatment or other manual handling, and produce a highly pure product, free of PCR inhibitors, that is less prone to degradation.

These new techniques respond to the demand for high-quality, highthroughput total RNA extraction methods posed by real-time quantitative PCR and other array-based assays.

Introduction

The ABI PRISM 6700 Automated Nucleic Acid Workstation is a flexible, fully automated system, designed to dramatically increase the throughput of nucleic acid preparation and reaction setup. In just over one hour, the 6700 workstation produces 96 purified samples of total RNA.

In less than 2.5 additional hours, the 6700 workstation prepares up to four fully assembled, 96-well reaction plates containing reagents, standards and controls for 5' nuclease assay analysis or other nucleic acid-based assays.

Chemistry

Applied Biosystems lysis chemistry uses a unique formulation to effectively lyse cells or tissues. The lysis solution is supplied as a 2X formulation and should always be used at a final concentration of 1X. Lysis occurs almost immediately after the cells are placed in the lysis buffer or a tissue sample is homogenized in it. The reagent inactivates cellular RNases, preventing degradation of the isolated nucleic acid, and keeps genomic DNA in solution, allowing a clean separation of RNA from all other cellular components.

The cellular lysate or tissue homogenate is transferred to a purification tray and the lysate or homogenate is passed through the tray under precisely controlled vacuum conditions. The 96-well tray (800 μL/well) contains a glass fibre m embrane which physically captures the RNA, a very low dead volume drip director, and an aerosol guard to prevent well-to-well cross-contamination. The RNA captured on the membrane is washed to remove any traces of cellular components and then re-solubilized and eluted from the membrane in a PCR compatible buffer.

Protocols

The purified total RNA can be used as follows: (Figure 1)

One or two daughter plates can be created at user-defined dilutions.

cDNA can be prepared from the purified RNA, using the 6700 workstations Peltier temperature control feature.

Up to four output plates can then be prepared, containing standards, controls, reagents for 5' nuclease assay or other nucleic acid-based assay reagents. A sample of the originally purified nucleic acid archive, dilution archive or cDNA archive, is added to the output plate.

The output plates are optionally heat-sealed with an optically clear film and are ready for analysis using the ABI PRISM 7700 or GeneAmp 5700 Sequence Detection System.

Archive tray covers are optionally placed on the purified nucleic acid archive, dilution archive or cDNA archive.

Definition of Terms

2X lysis reagent: Lysis Reagent, Nucleic Acid Purification (P/N 4305895)

lysis buffer: 1:1 mixture of P/N 4305895 and phosphate buffered saline, Ca2+/Mg2+ free

Methods

Lysis of Suspension or Adherent Cells Recommended input ranges for Applied Biosystems total RNA chemistry: 104-106 cells/well. Recommended maximum lysate volume for the 6700 workstation: 250 μL lysed cells/well in a 300 μL cell culture plate.*

(*See Ordering Information for recommended cell culture plates.)

Lysis and Purification of RNA from Suspension Cell C ultures

Important: Lysis reagent is formulated as a 2X solution. The final lysis buffer concentration in any lysis step should always be 1X for maximum RNA recovery.

Method 1 Automated lysis reagent addition by the 6700 workstation

1.Pellet suspension cells by centrifugation.

2.Remove the growth media by aspiration from all wells, taking care to ensure that the cell pellet is not disturbed.

3.Re-suspend cell pellet in 125 μL PBS (phosphate buffered saline, Ca2+/Mg2+ free).

4.Place the cell culture plate containing the cells in the primary input position of the 6700 workstation.

5.Activate the default lysis protocol (Figure 2).

6.Configure the lysis protocol to add 125 μL of 2X lysis reagent and to mix the sample at least 3 times after addition.

7.Activate the default RNA archive protocol to purify total RNA from the cell lysate (Figure 3).

8.If the default RNA archive protocol is used, purified RNA will be eluted in 150 μL of PCR compatible buffer.

Method 2 Manual lysis of suspension cells

1.Pellet suspension cells by centrifugation.

2.Remove the growth media by aspiration from all wells, taking care to ensure that the cell pellet is not disturbed.

3.Re-suspend cell pellet in 250 μL of a 1:1 mixture of lysis reagent and PBS (phosphate buffered saline, Ca2+/Mg2+ free).

4.Ensure complete nucleic acid release by taking up the lysate into the pipette tip at least three times, mixing the lysis buffer with the cell pellet.

5.Place the cell culture plate containing the lysate in the primary input position of the 6700 workstation.

6.Activate the default RNA archive protocol to purify total RNA from the cell lysate (Figure 3).

7.If the default RNA archive protocol is used, puri fied RNA will be eluted in 150 μL of PCR compatible buffer.

Note: If the cell culture growth media is Ca2+/Mg2+ free, lysis may be performed directly in the growth media by addition of an equal volume of 2X lysis reagent.

Lysis and Purification of RNA from Adherent Cell Cultures

Important: Lysis reagent is formulated as a 2X solution. The final lysis buffer concentration in any lysis step should always be 1X for maximum RNA recovery.

Method 1

Automated lysis reagent addition by the 6700 workstation

1.Remove the growth media by aspiration from all wells.

2.Re-suspend the cells in 125 μL PBS (phosphate buffered saline, Ca2+/Mg2+ free).

3.Place the cell culture plate containing the cells in the primary input position of the 6700 workstation.

4.Activate the default lysis protocol (Figure 2).

5.Configure the lysis protocol to add 125 μL of 2X lysis reagent and to mix the sample at least 3 times after addition.

6.Activate the default RNA archive protocol to purify total RNA from the cell lysate (Figure 3).

7.If the default RNA archive protocol is used, purified RNA will be eluted in 150 μL of PCR compatible buffer.

Method 2

Manual lysis of adherent cells

1.Remove the growth media by aspiration from all wells.

2.Re-suspend the cells in 250 μL of a 1:1 mixture of lysis reagent and PBS (phosphate buffered saline, Ca2+/Mg2+ free).

3.Ensure complete nucleic acid release by taking up the lysate into the pipette tip at least three times and thoroughly mixing the lysis buffer with the cells.

4.Place the cell culture plate containing the lysate in the primary input position of the 6700 workstation.

5.Activate the default RNA archive protocol to purify total RNA from the cell lysate (Figure 3).

6.If the default RNA Archive protocol is used, purified RNA will be eluted in 150 μL of PCR compatible buffer. Note: If the cell culture growth media is Ca2+/Mg2+ free, lysis may be performed directly in the growth media by addition of an equal volume of 2X lysis reagent.

Results

Applied Biosystems total RNA lysis chemistry is specified to give the following results when used as recommended.

Yield of Total RNA (RNase-free) Total RNA recovery: > 50% for seeding densities in the range of 104 and106 cells per well (Figure 4 and 5)

This recovery rate may be routinely assessed using the Cell Lysate Control Kit, containing lysates at two concentrations: 104 and 106 cells/well. Total RNA is purified from these two lysates and compared to RNA standards included in the kit in an ABI PRISM 7700 Sequence Detection System based RT+ 18S ribosomal RNA assay.

Precision and Reproducibility of Recovery Coefficient of variation (%CV): <30%

Total experimental coefficient of variation, including 6700 workstation purification, all pipetting steps and analysis by ABI PRISM 7700 Sequence Detection System is less than 30% (Figure 4 and 6).

Purity of Total RNA

Purity: < 0.5% w/w of genomic DNA

Demonstrated performance of A260/280 ratio: >1.9 (Figure 6 and 7)

Inhibition

CT suppression of neat sample: ≤0.5

The presence of inhibitors after purification of total RNA samples is a widespread problem with other total RNA extraction methods. The ABI PRISM 7700 Sequence Detection System and an RT+ GAPDH based assay easily detects the presence of such inhibitors. Applied Biosystems total RNA lysis reagents and purification technology readily removes them.

For very sensitive assessment of PCR performance, measure CT values on fourfold serial dilutions of an individual sample. To measure inhibition, plot a standard curve of the three most dilute samples and extrapolate to compare measured versus calculated CT values for the 1:4 and undiluted sample. PCR inhibitor is present if the measured CT value for the undiluted sample is suppressed by >0.5 cycles from the calculated CT value (Figure 8).

Cross-contamination Intra/inter-run, or well-to-well: <1 in 106 copies at an occurrence rate of ≤1 per tray

Range of Recovery

Figure 6 shows that for Raji cells, total RNA recovery versus cell input is linear for the range 10 pg100 μg, (101106 cells).

Caution: Input ranges >106 cells/well can cause blockages in the purification tray, which, in turn, causes overflow of the wash solution. This results in well-to-well cross-contamination and failure of the purification run (Figure 9).


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