|
PolyATtract® Automated System
The PolyATtract® Automated System builds on the innovative paramagnetic technology first introduced with the PolyATtract® mRNA Isolation Systems (mRNA direct from total RNA) and the PolyATtract® System 1000 (mRNA direct from cells or tissues). The PolyATtract® Automated System provides higher throughput mRNA isolation. The PolyATtract Automated System rapidly purifies mRNA from either total RNA or directly from cultured cells and tissue lysates. The system is designed for use with automated liquid-handling platforms like the Beckman Coulter Biomek® 2000 and FX. The system uses a biotinylated oligo(dT) primer to hybridize at high efficiency in solution to the 3’ poly(A)+ region present in most mature eukaryotic mRNAs. The hybrids are bound to streptavidin coupled to MagneSphere® Paramagnetic Particles, captured using a magnetic separation device, and washed at high stringency. The mRNA is eluted from the solid phase by the sample addition of ribonuclease-free, deionized water. The system removes >99% of the ribosomal RNA as judged by quantitative, real-time RT-PCR for 18S rRNA. No further processing of the purified RNA is necessary for end-point or quantitative, real-time RT-PCR analysis.
The PolyATtract® process is based on the efficient hybridization of a biotinylated oligo(dT) to poly(A)+ RNA in solution, rather than on the interaction of poly(A)+ RNA with oligo(dT) pre-bound to a particle. The hybrids are captured on the Streptavidin-MagneSphere® Paramagnetic Particles, washed at high stringency and finally poly(A)+ RNA is eluted in Nuclease-Free Water.
Figure 1. Schematic of the PolyATtract® Automated System protocol.
Benefits
Choice of starting material: Protocols for starting with total RNA, tissue lysates or cultured cells are provided. There is linear recovery of poly(A)+ RNA using 0.31–20µg of eukaryotic total RNA. Work with 0.02–5mg of eukaryotic tissue lysate or 102–106 cultured mammalian cells. Methods remove >99% of rRNA as judged by quantitative, real-time RT-PCR.
Retention of expression profile from total RNA: You don’t have to worry that you will lose mRNA species during purification. Expression profile of purified poly(A)+ RNA is equivalent to expression profile of purified total RNA from the same sample.
No cross-contamination of adjacent wells: No detectable well-to-well contamination as judged by end-point RT-PCR.
Compatible with many robotic platforms: Methods the Beckman Coulter Biomek® 2000 and Biomek® FX are available. The Technical Bulletin (#TB321) provides advice on adapting protocols to other robotic platforms.
The PolyATtract® Automated System provides linear recoveries of poly(A)+ RNA from 0.313ng to 20µg of total RNA.
Figure 2. Recovery of poly(A)+ RNA from total RNA. Serial dilutions of mouse liver total RNA were processed and analyzed for recovery of RNA using RiboGreen® (Molecular Probes). Data is the average of three isolations per sample. The average yield from 5µg of total RNA was 65.6±2.7ng with 1.31% recovery, there is a linear relationship between the input and recovered RNA.
The performance of purified poly(A)+ RNA in real-time RT-PCR was evaluated (Panel A). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message was amplified directly from total RNA, poly(A)+ mRNA purified from total RNA or poly(A)+ mRNA purified directly from cells. We found no significant differences in amplification between sources indicating little loss of purified message during the poly(A)+ isolation procedure. Additionally, we analyzed a dilution series using real-time RT-PCR to quantitate the removal of 18S ribosomal RNA during purification. 18S RNA was amplified from a serial dilution of total RNA and from purified poly(A)+ RNA. Comparison of the amplification profiles indicates that >99.5% of ribosomal RNA is removed (Panel B).
Figure 3. Real-time RT-PCR analysis of purified mRNA.
Aliquots of total RNA, mRNA isolated from total RNA, or mRNA isolated directly from 1 x 105 HeLa cells were reverse transcribed. Panel A. Five-microliter aliquots of the reverse transcription reactions were used for amplification of GAPDH target. Panel B. A dilution series of the total RNA reverse transcription reaction was compared to undiluted poly(A)+ RNA by amplification of an 18S ribosomal RNA target. While amplification of the GAPDH mRNA target is equivalent in the total and mRNA samples, dilution series analysis indicates that >99.5% of 18S rRNA was removed during purification. All reactions were performed using TaqMan® reagents from Applied Biosystems, Inc.
Additional Information
DNA and RNA Purification
View Online Catalog Information
PolyATtract® Automated System
PolyATtract and MagneSphere are registered trademarks of Promega Corporation.
Biomek is a registered trademark of Beckman Coulter, Inc.
TaqMan is a registered trademark of Roche Molecular Systems, Inc.
RiboGreen is a registered trademark of Molecular Probes, Inc.
|
