QIAamp RNA Blood Mini Kit

For purification of cellular RNA from fresh whole blood

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QIAamp RNA Blood Mini Kit (50)

Cat. No. / ID: 52304

For 50 RNA preps: 50 QIAamp Mini Spin Columns, 50 QIAshredder Spin Columns, Collection Tubes (1.5 ml and 2 ml), RNase-free reagents and buffers

KitBuffer

QIAamp RNA Blood Mini Kit

Buffer AW1

The QIAamp RNA Blood Mini Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

Rapid purification of high-quality, ready-to-use RNA
No organic extraction or alcohol precipitation
Consistent, high yields
Removal of contaminants and inhibitors

Product Details

The QIAamp RNA Blood Mini Kit provides silica-membrane-based purification of cellular RNA from up to 1.5 ml of fresh, whole human blood stabilized with any common anticoagulant, such as citrate, heparin, or EDTA. After homogenization using the QIAshredder spin column, a fast spin-column procedure simplifies RNA purification. Purification can be fully automated on the QIAcube Connect.

Performance

The QIAamp procedure completely removes RNases, contaminants, and enzyme inhibitors, yielding high-quality RNA suitable for any downstream application (see figures " High-quality RNA for northern analysis" and " Reliable RT-PCR analysis").

The QIAamp RNA Blood Mini Kit provides the highest-quality RNA with minimum copurification of DNA. However, as with any RNA purification method, some DNA contamination can be expected. For certain RNA applications that are sensitive to very small amounts of DNA, it may be necessary to remove any remaining DNA. In these cases, the QIAGEN RNase-Free DNase Set provides convenient on-column DNase treatment of RNA samples during QIAamp RNA procedures.

See figures

High-quality RNA for northern analysis.

Reliable RT-PCR analysis.

Principle

The QIAamp RNA Blood Mini Kit provides purification of cellular RNA using silica-membrane technology. No phenol–chloroform extraction is required. RNA binds specifically to the QIAamp silica-gel membrane while contaminants pass through. PCR inhibitors, such as divalent cations and proteins, are completely removed in two efficient wash steps, leaving pure RNA to be eluted in either water or a buffer provided with the kit.

QIAamp technology yields total cellular RNA from fresh whole blood and other sample sources that is ready to use in RT-PCR and blotting procedures. QIAamp sample preparation technology is fully licensed.

Procedure

The QIAamp RNA Blood Mini Kit simplifies isolation of RNA from blood with a fast spin-column procedure (see figure ""). Red blood cells are selectively lysed and white cells collected by centrifugation. White cells are then lysed using highly denaturing conditions which immediately inactivate RNases. After homogenization using the QIAshredder spin column, the sample is applied to the QIAamp spin column. Total RNA binds to the QIAamp membrane and contaminants are washed away, leaving pure RNA to be eluted in 30–100 µl RNase-free water (provided with the kit) for direct use in any downstream application.

See figures

QIAamp RNA Blood Mini procedure.

Applications

QIAamp RNA Blood Mini Kits provide a fast, easy method for the preparation of total cellular RNA from up to 1.5 ml of human whole blood. Contaminants and enzyme inhibitors, such as hemoglobin and heparin, are completely removed, leaving purified RNA ready for use in downstream applications, such as:

RT-PCR
Real-time RT-PCR
cDNA synthesis
Northern, dot and slot blotting
RNase/S1 nuclease protection
Differential display
Poly A+ RNA selection
Primer extension

In addition, QIAamp RNA Blood Mini Kits can be used to purify total RNA from tissues and cultured cells and to separate RNA from proteins, salt and other reaction components after enzymatic reactions, such as DNase digestions, proteinase digestions, RNA ligation and labeling reactions.

Supporting data and figures

QIAamp RNA Blood Mini procedure.

Specifications

Features	Specifications
Applications	PCR, real-time PCR, microarray
Elution volume	30–100 µl
Main sample type	Whole blood, tissue
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein	Cellular RNA
Sample amount	50–1.5 ml
Format	Spin columns
Processing	Manual (centrifugation)
Time per run or per prep	<1 hour
Technology	Silica technology
Yield	1–5 µg

Resources

For total RNA purification from human whole blood

Poster for download

Download Safety Data Sheets for QIAGEN product components.

FAQ

One milliliter of healthy human blood consists of cell types in approximately the following numbers:

Leucocytes (function: immune response) 4–7 x 106 cells
Thrombocytes (function: wound closing) 3–4 x 108 cells
Erythrocytes (function O2 and CO2 transport) 5 x 109 cells

FAQ ID -2951

Several kit options are available for this application. We recommend using the PAXgene Blood RNA System, which enables the collection, stabilization and transportation of 2.5 ml human whole blood samples, and subsequent rapid and efficient isolation of cellular RNA.

Other products for the isolation of RNA from whole human blood are the QIAamp RNA Blood Mini Kit and the RNeasy Midi Kit for processing up to 1.5 ml and 10 ml human whole blood, respectively.

FAQ ID -304

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

QIAGEN Proteinase K is a subtilisin-type protease, which cleaves at the carboxyl side of hydrophobic, aliphatic and aromatic amino acids. It is particularly suitable for short digestion times. It possesses a high specific activity over a wide range of temperatures and pH values with substantially increased activity at higher temperature. Soluble calcium is not essential for enzymatic activity. This means that EDTA, which is used to inhibit Mg2+-dependent enzymes such as nucleases, will not inhibit Proteinase K activity.

QIAGEN Protease is a broad-specificity Serine protease with high activity, cleaving preferentially at neutral and acidic residues. It is an economical alternative to Proteinase K for isolation of native DNA and RNA from a variety of samples.

For more information on activity of QIAGEN Protease and Proteinase K in various buffers please click here.

FAQ ID -761

Yes. The RNeasy MinElute Cleanup Kit has been developed specifically for cleaning up and concentrating RNA samples. You can also follow the protocols for RNA cleanup in the RNeasy Mini and RNeasy Midi/Maxi Handbook.

FAQ ID -286

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

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

According to an Interview with Professor Dennis Lo published in QIAGEN News Molecular Diagnostics, Issue No. 5, 2002, healthy individuals have about 500-1000 genome equivalents (DNA) per ml serum/plasma.

For free-circulating DNA in plasma, the concentration can range from 1–100 ng/ml in healthy individuals.

FAQ ID -635

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

The A₂₆₀/ A₂₈₀ ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A₂₆₀/A₂₈₀ ratio can vary greatly. Lower pH results in a lower A₂₆₀/ A₂₈₀ ratio and a reduced sensitivity to protein contamination*.

For accurate values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer with the same solution. Please see the Appendix sections in the RNeasy handbooks for additional information.

* Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.

FAQ ID -97

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

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