RNeasy 96 BioRobot Kits

For 96-well purification of total RNA from cells or any tissue on the BioRobot Universal System

Features

  • High yields of total RNA from cells and all tissues
  • Fast and convenient sample processing
  • Pure, high-quality RNA without phenol contamination
  • High-performance RNA for all downstream applications
RNeasy 96 Universal Tissue 8000 Kit (12)

Cat. No. / ID: 967852

For 12 x 96 total RNA preps on the BioRobot 8000 or BioRobot Universal System: 12 RNeasy 96 Plates, Collection Microtubes, Elution Microtubes CL, Caps, S-Blocks, QIAzol Lysis Reagent, RNase-Free Reagents and Buffers
¥571,000
Sample type
Tissue
Cells
RNeasy 96 BioRobot Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

RNeasy 96 BioRobot Kits enable total RNA purification in 96-well format for BioRobot instruments from cultured cell samples and any tissue. Tissues are efficiently lysed and homogenized in QIAzol Lysis Reagent while cells are lyses in Buffer RLT. High-quality RNA is purified from tissues and cells using silica-membrane RNeasy 96 plates. Automated purification is performed using the BioRobot Universal System, which processes up to 96 or 192 samples per run followed by real-time RT-PCR setup. Tissue samples can be conveniently stabilized using Allprotect Tissue Reagent, and efficiently disrupted using the TissueLyser II. Nonfatty tissues can be stabilized using the RNAprotect Tissue Reagent.

Performance

The RNeasy 96 system provides fast and reproducible total RNA purification in 96-well format for BioRobot instruments for high-throughput gene expression profiling. The RNA is suitable for sensitive applications such as quantitative, real-time RT-PCR and microarray analysis (see figure " High-quality RNA for sensitive analysis of a low-copy transcript"). Sample sizes range from 10 to 5 x 105 cells (see figure " RT-PCR of RNA from as few as 100 cells"), and high-quality RNA can be purified from large numbers of samples (see figure " Reproducible yields of high-quality RNA"). 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% using the QuantiTect Probe RT-PCR Kit (see figure " Repeatability of fully automated RNA purification").


Compared with standard silica-membrane procedures, the RNeasy 96 system provides higher yields of total RNA for all tissue types (see table “Typical total RNA yields using the RNeasy 96 Universal Tissue Kit”).

Typical total RNA yields using the RNeasy 96 Universal Tissue Kit

Tissue RNA yield* ((µg per 10 mg of tissue)
Kidney 5–40
Liver 15–80
Lung 5–15
Heart 5–25
Muscle 5–35
Brain 5–20
Adipose tissue 0.5–2.5
Spleen 15–100
Intestine 10–60
Skin 2–5

High-quality RNA can be isolated from tissues preserved either by freezing or by stabilization with RNAprotect at room temperature (see figure " High-quality total RNA from rat liver and muscle tissue"). Intact RNA purified from difficult-to-lyse fibrous and fatty tissues is suitable for downstream applications (see figures " High-quality RNA from skin /  adipose /  liver /  intestine tissue" and " Real-time analysis of high-quality RNA from rat brain").

See figures

Principle

The RNeasy 96 BioRobot 8000 Kit provides a standardized, fast, reliable method for isolation of high-quality RNA from large numbers of samples (see table “Specifications of the RNeasy 96 BioRobot 8000 Kit”). The simple procedure is key to the speed and efficiency of RNeasy 96 technology. 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 (see figure " RNeasy 96-well plate"), RNA binds to the silica 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. Since the RNeasy procedure enriches for RNA species >200 nt, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs.

Specifications of the RNeasy 96 BioRobot 8000 Kit

Specification Details
Format RNeasy 96-well plates with 1.2 ml collection microtubes
Sample source Animal or human cells
Sample size 10–5 x 105 cells
Binding capacity Up to 100 µg RNA per well
Yield* from 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 45–140 µl
See figures

Procedure

Fully automated total RNA isolation using the RNeasy 96 BioRobot 8000 Kit is performed on the BioRobot 8000 workstation or BioRobot Universal System. The BioRobot worktable has the 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 – 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. RNA purification can also be performed manually on the QIAvac 96 vacuum manifold and/or the 96-Well-Plate Centrifugation System using the RNeasy 96 Kit.


The RNeasy 96 Universal Tissue 8000 Kit integrates efficient phenol/guanidine-based lysis and silica-membrane purification with the speed of vacuum processing. Following homogenization and phase separation, the BioRobot 8000 workstation or BioRobot Universal System provides walkaway automation of the procedure, for total RNA purification from all types of animal tissue (see flowchart " RNeasy 96 Universal Tissue 8000 procedure").


Tissue is first efficiently lysed using QIAzol Lysis Reagent and the TissueLyser II. This provides rapid and parallel disruption of tissues and inactivation of RNases to ensure purification of intact RNA. After phase separation by centrifugation, the BioRobot workstation removes the aqueous phase and adds ethanol to provide appropriate binding conditions. The workstation then applies the sample to the wells of the RNeasy 96 Universal Tissue plate, where total RNA binds and contaminants are efficiently washed away. High-performance RNA is then eluted in a small volume of water, ready for use in any downstream application.

See figures

Applications

The RNeasy 96 BioRobot 8000 Kit consistently provides the highest-quality RNA for:

  • High-throughput RNA applications in gene expression analysis
  • Sensitive applications such as quantitative, real-time RT-PCR
  • Pharmacological and toxicological research

The combination of QIAzol and RNeasy technologies results in highly pure RNA without phenol carryover. RNA purified using the RNeasy 96 Universal Tissue 8000 Kit is suitable for all downstream applications, including real-time RT-PCR.

Comparison of RNeasy 96 BioRobot Kits

Features RNeasy 96 BioRobot 8000 Kit RNeasy 96 Universal Tissue 8000 Kit
Applications PCR, real-time PCR, microarray PCR, qPCR, real-time PCR
Elution volume 45–140 µl Custom
For automated processing

BioRobot 8000 Workstation

or BioRobot Universal System

BioRobot 8000 Workstation

or BioRobot Universal System

Format 96-well plate 96-well plate
Main sample type Cultured cells Tissue samples
Processing Automated Automated

Purification of total RNA, miRNA,

poly A+ mRNA, DNA or protein

RNA RNA
Sample amount 5 x 105 20–80 mg
Technology Silica technology Silica technology
Yield 1.3–3.1 µg Varies

Supporting data and figures

Resources

Kit Handbooks (3)
For high-throughput RNA purification from all types of animal tissue, automated on the BioRobot Universal System
For high-throughput RNA purification from animal and human cells, automated on the BioRobot Universal System, BioRobot Gene Expression — Real-Time RT-PCR, or BioRobot 8000

FAQ

What sample types can be used with the RNeasy Plus 96 Kit?

The RNeasy Plus 96 Kit provides high-throughput purification of total RNA from cultured cells grown in 96-well plates. There are protocols for small amounts of easy-to-lyse tissues, allowing RNA purification without the use of phenol using spin technology or vacuum/spin technology.  However, for most applications, we recommend the RNeasy 96 Universal Tissue Kit, which uses phenol-based sample lysis to enable RNA purification from all types of tissue.

 

FAQ ID -1991
I accidentally stored Buffer RDD of the RNase-Free DNase Set at°C. Will it still function?
Yes, buffer RDD of the RNase-Free DNase Set will still work. Please make sure that the buffer is thawed completely without any precipitates before using it. If precipitates are visible, the buffer should be slightly heated.
-20
Do you have a kit for RNA isolation from any kind of sample type?

The RNeasy 96 Universal Tissue Kit enables high-throughput purification of RNA from any animal or human tissue sample, including difficult-to-lyse fibrous and fatty tissues. For single tube format RNA purification, the RNeasy Plus Universal Kit is also available. 

Please refer to the Selection guide for RNA isolation for all sample types to find the optimal solution for your sample source.

FAQ ID -627
What is the composition of Buffer RLT?

The exact composition of Buffer RLT is confidential. This buffer is a proprietary component of RNeasy Kits. Buffer RLT contains a high concentration of guanidine isothiocycanate, which supports the binding of RNA to the silica membrane. Buffer RLT can be purchased separately (cat. no. 79216)

Note: note that ß-mercaptoethanol should be added to Buffer RLT before use to effectively inactivate RNAses in the lysate (10 µl ß-Mercaptoethanol per 1 ml Buffer RLT).

FAQ ID -2793
How can I check the integrity of RNA purified using RNeasy Kits?

The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by denaturing-agarose gel electrophoresis, the Agilent 2100 bioanalyzer, or the QIAxcel Advanced System with the QIAxcel RNA QC Kit v2.0.

 

The respective ribosomal species should appear as sharp bands on the stained gel. 28S ribosomal RNA bands should be present with an intensity approximately twice that of the 18S RNA band. If the ribosomal bands are not sharp, but appear as a smear of smaller sized RNAs, it is likely that the RNA sample has suffered major degradation during preparation.

 

Size of ribosomal RNAs from various sources

 

Source rRNA Size (kb) E. coli 16S 1.5   23S 2.9 S. cerevisiae 18S 2.0   26S 3.8 Mouse 18S 1.9   28S 4.7 Human 18S 1.9   28S 5.0

 

 

 

 

 

 

 

 

 

FAQ ID -1024
How can I check for purity of RNA isolated using RNeasy Kits?

Purity of RNA isolated with RNeasy Kits can be evaluated by determining the ratio of absorbance readings at 260 nm and 280 nm (A260/A280). This ratio provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV range, such as protein.

Note that the A260/A280 ratio is influenced considerably by pH. As water is unbuffered, the pH and the resulting 260/280 ratio can vary greatly. For an accurate determination of purity, we recommend measuring the 260/280 absorbance in 10 mM Tris-Cl, pH 7.5. Be sure to calibrate the spectrophotometer with the same solution. Pure RNA has an A260/A280 ratio of 1.9-2.1. However, values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris, pH 7.5) with some spectrophotometers.

For details on how the pH influences nucleic acid purity measurements, please review the reference 'Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity', by Wilfinger WW, Mackey K, Chomczynski P, Biotechniques. 1997 Mar;22(3):474-6, 478-81.

FAQ ID -1023
I ran my RNA out on an agarose gel and can see lots of bands similar to a ladder. Why?

RNA has a high degree of secondary structure that needs to be resolved or denatured before running the sample out on a gel. A formaldehyde gel needs to be used to disrupt the secondary structure and eliminate a ladder effect. For details please refer to the chapter "A Guide to Analytical Gels" in the QIAGEN Bench Guide.

Some banding pattern may remain due to the presence of mRNA transcripts of different lengths specific for the respective cell or tissue type.

FAQ ID -745
What is the difference between disruption and homogenization in the RNeasy System?

Disruption:

Complete disruption of cell walls and plasma membranes of cells and organelles is absolutely required to release all RNA contained in a sample. Different samples require different methods to achieve complete disruption. Please refer to the section 'Disruption and homogenization of starting materials' in the RNeasy Mini Handbook. Incomplete disruption results in significantly reduced RNA yields.

Homogenization:

Homogenization is necessary to reduce the viscosity of the cell lysates produced by disruption. Homogenization shears the high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in genomic DNA contamination, and inefficient binding of RNA to the RNeasy membrane resulting in reduced yields.

FAQ ID -139
Can the RNase-Free DNase Set be used for DNase digestions of RNA in solution?

Yes. Even though buffer RDD in the RNase-Free DNase Set is optimized for on-column DNase digestion, the buffer is also well-suited for efficient DNase digestion in solution. Please note that the reaction must be cleaned up after the off-column DNase digest to remove the enzyme and buffer RDD, which will interfere with subsequent RT reactions.

A protocol for in-solution DNase digestion using the RNase-Free DNase Set can be found in Appendix C of the RNeasy MinElute Cleanup Handbook. For subsequent RNA Cleanup, use either the RNeasy MinElute Cleanup Kit, or follow the instructions for RNA Cleanup in the RNeasy Mini Handbook.

 

FAQ ID -619
How should RNeasy Kits be stored and how long are they stable?
RNeasy Mini, Midi and Maxi Kits should be stored dry at room temperature (15 to 25°C). The RNeasy MinElute Spin Columns of the RNeasy Micro Kit and RNeasy MinElute Cleanup Kit should be stored at 4°C. RNeasy Kits are stable for at least 9 months under these conditions.
FAQ ID -103
Can I buy the RNeasy Mini columns, RNeasy MinElute columns, RNeasy Midi columns or the RNeasy Maxi columns separately?

We do not sell the RNeasy MinElute columns, RNeasy Mini columns, RNeasy Midi columns or the RNeasy Maxi columns separately.

In general, we always provide extra volume of buffers in our kits to account for pipetting errors and such. If you are left with extra buffers after using up all the columns in a kit, please refer to the Material Safety Data Sheet for respective kit to dispose off any unused buffers.

FAQ ID - 3388
What can be used as an alternative to the A260 measurement for quantification of small amounts of RNA and DNA?

Small amounts of RNA and DNA may be difficult to measure spectrophotometrically. Fluorometric measurements, or quantitative RT-PCR and PCR are more sensitive and accurate methods to quantify low amounts of RNA or DNA.

Fluorometric measurements are carried out using nucleic acid binding dyes, such as RiboGreen® RNA Quantitation Reagent for RNA, and PicoGreen® DNA Quantitation Reagent for DNA (Molecular Probes, Inc.).

FAQ ID -728
How do you ensure that RNeasy buffers are RNase-free?

Our RNeasy buffers are subjected to stringent quality-control tests to ensure that they are indeed RNase-free. Buffer RPE concentrate and RNase-free water are tested for absence of RNases by incubating 4 µg of total HeLa-RNA in these solutions for 3 hours at 37°C, followed by monitoring RNA integrity via denaturing agarose gel electrophoresis and ethidium bromide staining.

Buffer RLT and Buffer RW1 are inherently RNase-free, since the buffers themselves inactivate RNases during the RNeasy procedure.

FAQ ID -113
Effects of low A260/A230 ratios in RNA preparations on downstream applications

The efficiency of downstream applications depends strongly on the purity of the RNA sample used.  Pure RNA should yield an A260/A230 ratio of around 2 or slightly above; however, there is no consensus on the acceptable lower limit of this ratio.  Possible candidates that can increase the A230 include “salt”, carbohydrates, peptides, and phenol (or aromatic compounds in general).  In our experience, the increased absorbance at 230 nm in RNA samples is almost always due to contamination with guanidine thiocyanate, present at very high concentrations in the lysis buffer or extraction reagent used in most RNA purification procedures.

Please find an article discussing the effect of low 260/230 ratios in RNA preparations on downstream applications on page 7 of QIAGEN Newsletter March 15, 2010 . In summary, we found that concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of downstream applications.

 

 

FAQ ID -2248
What has to be done to an RNA sample before loading it onto an Agilent Bioanalyzer?

For RNA isolated on the BioRobot EZ1 and BioRobot M48:

The RNA can be directly applied to the Agilent Bioanalyzer, since it is being denatured in the final protocol steps of these isolation procedures.

For RNA prepared with all other QIAGEN RNA Isolation Products:

We recommend to denature the samples in a water bath for 2 min at 70°C, and then place them directly on ice prior to loading them onto the Agilent Bioanalyzer.

 

FAQ ID -528
How do I safely inactivate biohazardous flow-through material?

Always dispose of potentially biohazardous solutions according to your institution’s waste-disposal guidelines. Although the lysis and binding buffers in QIAamp, DNeasy, and RNeasy kits contain chaotropic agents that can inactivate some biohazardous material, local regulations dictate the proper way to dispose of biohazards. DO NOT add bleach or acidic solutions directly to the sample-preparation waste. Guanidine hydrochloride in the sample-preparation waste can form highly reactive compounds when combined with bleach.
Please access our Material Safety Data Sheets (MSDS) online for detailed information on the reagents for each respective kit.

FAQ ID -12
What is the composition of Buffer RPE?
The exact composition of Buffer RPE is confidential. Buffer RPE is a mild washing buffer, and a proprietary component of RNeasy Kits. Its main function is to remove traces of salts, which are still on the column due to buffers used earlier in the protocol. Ethanol, which is added by the user just before using the kit for the first time, is an important ingredient of Buffer RPE.
FAQ ID -2797
What is the composition of Buffer RW1?

The exact composition of Buffer RW1 is confidential. Buffer RW1 is a proprietary component of RNeasy Kits. Buffer RW1 contains a guanidine salt, as well as ethanol, and is used as a stringent washing buffer that efficiently removes biomolecules such as carbohydrates, proteins, fatty acids etc., that are non-specifically bound to the silica membrane. At the same time, RNA molecules larger than 200 bases remain bound to the column.

Note: Buffer RW1 should not be used for isolation of small RNAs, for example, microRNAs or fragmented RNA from formalin-fixed tissues, as these smaller fragments will be washed away.  Buffer RWT should be used instead.

FAQ ID -2796
How should I quantify RNA isolated with RNeasy Kits?

The concentration of RNA isolated with RNeasy Kits can be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. Absorbance readings should be greater than 0.15 to ensure significance. An absorbance of 1 unit at 260 nm corresponds to 40 µg of RNA per ml (A260 = 1 = 40 µg/ml). This relationship is valid for measurements in water. Therefore, dilute RNA in water to quantify it spectrophotometrically.

An example of the calculations involved in RNA quantification is shown below. Use the buffer in which the RNA is diluted to zero the spectrophotometer:

  • Volume of RNA sample = 100 µl
  • Dilution = 10 µl of RNA sample + 490 µl distilled water (1/50 dilution)
  • Absorbance of diluted sample measured in a 1 ml cuvette (RNase-free): A260 = 0.23
  • Concentration of original RNA sample = 40 x A260 x dilution factor = 40 x 0.23 x 50
  • RNA concentration: 460 µg/ml

  • Total yield = concentration x volume of sample (ml) = 460 µg/ml x 0.1 ml
  • RNA yield: 46 µg

For additional information on RNA quantitation and handling, see the Appendix section in the RNeasy Mini Handbook.

FAQ ID -32