Format:
Sample source:
Sample size:
Binding capacity:
Yield (1 x 105 cells)*
Elution volume:
Further information:
RNeasy Stabilization and Total RNA
Isolation System
For automated or manual high-throughput RNA minipreparation from cells
|
Format: |
RNeasy 96-well plates with 1.2 ml collection microtubes; automated or manual | |
|
Sample source: |
Animal or human cells,
in vitro transcripts, or enzymatic reactions |
|
|
Sample size: |
1 ¨C 5 x 105 cells | |
|
Binding capacity: |
Up to 100 µg RNA per well | |
|
Yield (1 x 105 cells)* |
||
| HeLa: | 1.6 µg | |
| LMH: | 1.3 µg | |
| COS-7: | 3.1 µg | |
| Huh7 | 2.0 µg | |
| Jurkat: | 1.4 µg | |
|
Elution volume: |
45¨C140 µl | |
* Amounts can vary due to developmental stage, growth conditions used, etc. Since the RNeasy procedure enriches for RNA species >200 nt, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs.
The RNeasy 96 system provides a standardized, reliable method for isolation of high-quality RNA from large numbers of samples. Sample sizes range from 1 to 5 x 105 cells. The system provides fast and reproducible total RNA purification for high-throughput gene expression profiling using sensitive applications such as quantitative real-time RT-PCR and microarray analysis. Individual variations are low throughout the entire purification process; TaqMan threshold-cycle values are easily achieved at the end of the process with a coefficient of variation (CV) less than 3% (see figure "Repeatability of Fully Automated RNA Purification and TaqMan RT-PCR Setup").
Reproducible Yields of High-Quality RNA with RNeasy 96 Kit
|
|
A Agarose gel analysis. HeLa cells (5 x 105) from a homogeneous cell culture were pelleted in each well of a 96-well cell-culture plate and the total RNA isolated using the RNeasy 96 system. Half of each eluate was analyzed on a formaldehyde agarose gel. B The corresponding northern blot, hybridized with a 32P-labeled GAPDH probe. C Analysis of RNA yields. Each square represents the yield from a preparation originating from one well of the 96-well cell-culture plate. |
The key to the speed and efficiency of RNeasy 96 technology is the simple procedure. Cells can be grown and directly lysed in 96-well cell-culture dishes. After transfer of the lysates to the wells of the RNeasy 96 plate, RNA binds to the silica-gel membrane in each well, and contaminants are washed away. Pure RNA is then eluted in RNase-free water into individual collection tubes suitable for long-term storage and is ready to use for any experiment.
The RNeasy 96 Kit includes protocols
for total RNA isolation, cytoplasmic RNA isolation*, and RNA cleanup†.
RNeasy 96 plates can be processed on:
RNA isolation can be semi-automated on the BioRobot 9604 workstation or fully automated on the BioRobot 8000 workstation.
The BioRobot 8000 worktable has capacity to purify RNA from up to 192 samples in a single run. With the one-plate protocol, the system can carry out both RNA purification and reaction setup in a single run ¡ª from living cell cultures in 96-well plates, to RT-PCR mixtures, ready to use in real-time gene expression analysis.
Filter tips prevent cross-contamination, and precision robotic handling provides minimal well-to-well variation and high repeatability. Highly sensitive results are achieved in real-time RT-PCR using the QuantiTect Probe RT-PCR Kit with QIAGEN Operon primers and fluorescent probes (see figure "Repeatability of Fully Automated RNA Purification and TaqMan RT-PCR Setup").
* Manual and Biorobot 9604 procedure
only
† Manual procedure only
²Ù×÷ÊÖ²á:
RNeasy 96 Handbook 01/02 (PDF version, 1560 KB)
RNeasy 96 BioRobot 9604 Handbook 01/02 (PDF version, 790 KB)
RNeasy 96 BioRobot 8000 Handbook 04/02 (PDF version, 271 KB)
QIAvac Handbook 09/01 (PDF version, 1615 KB)
High-throughput RNA analysis is an increasingly important tool in biomedical research, diagnostics, and drug discovery. The lack of an efficient, high-throughput method has made RNA isolation the limiting step until now.
The RNeasy 96 Kits consistently provide the highest-quality RNA for any application, ideal for high-throughput drug screening, pharmacological research, and molecular diagnostics. Isolated RNA is suitable for sensitive applications such as quantitative RT-PCR, and TaqMan (see figure "High-Quality RNA for Sensitive Analysis of a Low-Copy Transcript") and LightCycler analysis.
High-Quality RNA for Sensitive Analysis of a Low-Copy Transcript
|
|
RNA was isolated from 1 to 1 x 105 HeLa S3 cells using the RNeasy 96 BioRobot 8000 procedure. Total RNA was eluted in 100 µl RNase-free water, and 5 µl was used for RT-PCR. Quantitative, real-time, one-step RT-PCR analysis was carried out on an ABI Sequence Detection System using the QuantiTect Probe RT-PCR Kit with primers and probe specific for the low-copy c-fos transcript. A Amplification plot B CT values. Error bars represent standard deviation from 8 different samples for each cell number. |
RT-PCR of RNA from ¡Ý100 Cells
 |
RT-PCR of cytoplasmic RNA isolated with the RNeasy 96 Kit from the indicated numbers of HeLa cells. Ten µl (1/10) of the eluate was reverse-transcribed with oligo-dT primer and 5 µl (1/5) of the cDNA mix was used in a 100 µl PCR in which a 214 bp fragment of beta-actin was amplified. C¨C: no template; C+: beta-actin in vitro transcript; M: 100 bp DNA ladder. |
Repeatability of Fully Automated RNA Purification and TaqMan RT-PCR Setup
 |
RNA was isolated from 96 aliquots (5 x 104 cells each) of a HeLa S3 cell culture using the RNeasy 96 BioRobot 8000 procedure. Quantitative, real-time, one-step RT-PCR was set up in the same protocol on the BioRobot 8000 workstation, using the QuantiTect Probe RT-PCR Kit with QIAGEN Operon primers and dual-labeled probe specific for the low-copy c-myc transcript. Threshold cycles (CT) are shown for all 96 samples. The mean CT was 21.34 ¡À 0.34 (mean ¡À standard deviation), representing a CV of 1.6%. |
|
* Requires use of either QIAvac 96 or the QIAGEN 96-Well-Plate Centrifugation System. |
