Navigation Links
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 membrane 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 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.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, 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.

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).


'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
2. High-Throughput Isolation of Total RNA
3. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
4. Fast Isolation of Total RNA from Small Numbers of Cells
5. Total RNA Isolation Protocol 1,2,3
6. Isolate It All: siRNA miRNA Total RNA Native Protein
7. Isolate Total RNA and Protein From the Same Sample
8. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
9. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
10. Total RNA from Whole Blood for Expression Profiling
11. RNAPrep - Total RNA Isolation Purification
Post Your Comments:
(Date:1/15/2014)... January 15, 2014 The Microcompetition with Foreign ... disease. One of these latent viruses is the Epstein Barr ... (RA). Rheumatoid arthritis (RA) is a chronic inflammatory disease that ... study found that RA patients have high concentrations of EBV ...
(Date:1/15/2014)... Sunnyvale, CA (PRWEB) January 15, 2014 ... throughput research solutions, today announced that Lupin Limited, one ... of Freeslate’s CM Protégé PharmD System for ... in Mumbai, India, is focused on a wide range ...
(Date:1/14/2014)... Date: Friday, April 11, 2014 , Time: 6 p.m. ... Road, Warrington, Pa. , Details: The Hepatitis B ... a cure for hepatitis B and improving the quality of ... Ball on Friday, April 11 at Warrington Country Club in ...
(Date:1/14/2014)... provider of strategic communications services to corporations and organizations preparing for ... United States and Europe , today ... to the firm,s Washington, D.C. office. ... years of service as Associate Commissioner for the Office of External ...
Breaking Biology Technology:Study: Rheumatoid Arthritis (RA) Patients Have EBV; The CBCD Says this is Consistent with Microcompetition 2Study: Rheumatoid Arthritis (RA) Patients Have EBV; The CBCD Says this is Consistent with Microcompetition 3Lupin Selects Freeslate’s CM Protégé PharmD System to Accelerate Polymorph Screening for Drug Development 2Lupin Selects Freeslate’s CM Protégé PharmD System to Accelerate Polymorph Screening for Drug Development 3Hepatitis B Foundation to Host Annual Crystal Ball Gala 2Former FDA Associate Commissioner Returns To 3D Communications 2
... VANCOUVER, June 6 /PRNewswire-FirstCall/ - QLT Inc. (NASDAQ: ... completed its review of QLT,USA, Inc.,s labeling supplement ... (G6PD) screening and blood monitoring,requirements., "We are ... remove,the need for blood monitoring from the Aczone ...
... Based on Increased 5-Year Survival Data for Responding ... Genta,Incorporated (OTC Bulletin Board: GNTA) announced that the ... Drug Administration (FDA) for its,New Drug Application (NDA) ... with relapsed or refractory chronic,lymphocytic leukemia (CLL). The ...
... from AHIMA, CHICAGO, June 5 "AHIMA,s Board ... promises to strengthen the effort,to address health IT from ... for senior executives. In addressing the need to,realize real ... critical,leadership challenge. AHIMA will continue working to support the ...
Cached Biology Technology:QLT announces positive Health Canada decision on Aczone(R) 2Genta Submits NDA Amendment to FDA for Genasense as Treatment of Chronic Lymphocytic Leukemia 2Genta Submits NDA Amendment to FDA for Genasense as Treatment of Chronic Lymphocytic Leukemia 3Genta Submits NDA Amendment to FDA for Genasense as Treatment of Chronic Lymphocytic Leukemia 4
(Date:7/9/2014)... Atlas (TCGA) Research Network have identified novel mutations ... the most common subtype of lung cancer. Knowledge ... of possible therapeutic targets for this disease and ... treatable mutations because many potent cancer drugs that ... jointly funded and managed by the National Cancer ...
(Date:7/9/2014)... that can attack soybean crops, why would charcoal rot ... University of Illinois scientists cite the earth,s changing climate ... the fungus that causes charcoal rot. , Fungi may ... Macrophomina phaseolina , the fungus that causes charcoal ... the climate continues to change and we see more ...
(Date:7/9/2014)... with pain and fever but often can be defeated with ... and hard to beat. Now, scientists have built a new ... pyramids. Published in the journal ACS Applied Materials & ... and kill more of them than medicine alone. , David ... can lie in wait, undetectable in the human body or ...
Breaking Biology News(10 mins):Study identifies novel genomic changes in the most common type of lung cancer 2Study identifies novel genomic changes in the most common type of lung cancer 3Climate change provides good growing conditions for charcoal rot in soybeans 2Climate change provides good growing conditions for charcoal rot in soybeans 3
... North Hollywood, CA August 13, 2009 - The ... and support for myeloma patients, families, researchers and physicianstoday ... link between environmental toxins and bone disease in multiple ... of cells in the bone marrow that affect production ...
... the splendidly colorful leaves adorning the trees are a delight ... yellow, while the United States and East Asia boast lustrous ... differences in autumnal hues around the world? A new theory ... Education- Biology at the University of Haifa-Oranim and Prof. Jarmo ...
... deaths has declined steadily in the last three decades. ... age groups have shown some improvement, according to a ... of the American Association for Cancer Research. ... better treatment, have resulted in profound gains, but these ...
Cached Biology News:New study suggests possible genetic links between environmental toxins and multiple myeloma 2Why are autumn leaves red in America and yellow in Europe? 2Why are autumn leaves red in America and yellow in Europe? 3Cancer mortality rates experience steady decline 2
...
These 18x18 square No.1 Corning cover glasses are made from No. 0211 zinc titania glass and are 0.16 to 0.19mm thick....
...
Experion spin filters are used to filter the gel matrix and gel stain solution prior to use with the Experion automated electrophoresis system. Supplied as 10 spin filters....
Biology Products: