RNeasy Kits

For purification of total RNA from cells, tissues, and yeast

Features

  • Fast procedure delivering high-quality total RNA in minutes
  • Ready-to-use RNA for high performance in any downstream application
  • Consistent RNA yields from small to large amounts of starting material
  • No phenol/chloroform extraction, no CsCl gradients, no LiCl or ethanol precipitation
  • High-throughput processing in 96-well format and automatable protocols
RNeasy Micro Kit (50)

Cat. No. / ID: 74004

50 RNeasy MinElute Spin Columns, Collection Tubes (1.5 ml and 2 ml), RNase-free DNase I, Carrier RNA, RNase-free Reagents and Buffers
$538.00
Column typePlate type
Micro
Mini
Mini QIAcube
Midi
Maxi
96 well
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RNeasy Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

RNeasy Kits are the Gold standard for total RNA isolation. They provide fast purification of high-quality RNA from small to large amounts of cells, tissues, and yeast using silica-membrane RNeasy spin columns or 96-well plates. Tissue samples can be conveniently stabilized using RNAprotect Tissue Reagent or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor II or TissueLyser II or LT system. The RNeasy 96 Kit enables high-throughput purification of total RNA from up to 96 cultured-cell samples using silica-membrane RNeasy 96 plates. A dedicated RNeasy QIAcube Kit enables automated purification of 1–12 samples on the QIAcube Connect.

Performance

RNeasy Kits deliver highly reproducible yields of total RNA from small to large samples. Total RNA can reliably be purified from small numbers of cells, including a single cell, as well as from small amounts of standard tissues (see figures  "Reliable RNA isolation from a single cell",  "Highly reproducible yields for sensitive applications" and  "High-quality total RNA from fine needle aspirates") using the RNeasy Micro Kit. RNA purified using the RNeasy Micro Kit provides maximum sensitivity with precious samples in quantitative gene expression analyses, such as real-time RT-PCR, by efficient on-column digestion of genomic DNA (see figure  "Efficient on-column removal of genomic DNA").
Total RNA is easily purified from animal or human cells, animal or human tissues, and yeast (see table “Total RNA yields obtained with RNeasy Kits” and figure  "RT-PCR of RNA from as few as 100 cells").

Total RNA yields obtained with RNeasy Kits

Source Starting material Average yield
Animal cells Micro Mini Midi Maxi 96-well Micro Mini Midi Maxi c
LMH 5 x 105 1 x 106 7 x 107 5 x 108 1 x 105 12 µg 850 µg 5.7 mg 1.3 µg*
HeLa 5 x 105 1 x 106 7 x 107 4 x 108 1 x 105 5mg 15 µg 1000 µg 6.0 mg 1.6 µg*
COS-7 5 x 105 1 x 106 3 x 107 1.8 x 108 1 x 105 17.5 mg 35 µg 950 µg 5.8 mg 3.1 µg*
Lymphocytes
(unstimulated)
5 x 105 1 x 106 1 x 108 5 x 108 0.5 µg 50 µg 0.3 mg
Huh7 5 x 105 1 x 105 7.5mg 2.0 µg*
Jurkat 5 x 105 1 x 105 1.4 µg*
Mouse tissue  
Liver 5 mg 10 mg 200 mg 1 g 15 µg 40 µg 700 µg 3.6 mg
Lung 5 mg 10 mg 100 mg 0.5 g 5 µg 10 µg 130 µg 0.6 mg
Spleen 5 mg 10 mg 200 mg 1 g 15 µg 35 µg 600 µg 3.2 mg
Yeast cells  
S. cerevisiae 1 x 107 2 x 108 1 x 109 25 µg 450 µg 2.4 mg

Total RNA purified with the RNeasy Maxi Kit is of high quality and is suitable for many downstream applications (see figure  "High-quality RNA from a variety of samples"). Total RNA is easily purified with the RNeasy Maxi Kit from large amounts of starting material including animal or human cells, animal or human tissues, and yeast cells (see table “Total RNA yields obtained with RNeasy Kits”).

The RNeasy 96 system provides fast and reproducible total RNA purification 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").

See figures

Principle

RNeasy Kits allow efficient purification of total RNA from small to large amounts of starting material, including samples such as microdissected tissues and fine needle aspirates, from milligram amounts of fibrous tissues including heart and muscle tissue, and from small numbers of cells down to single cells (e.g., FACS sorted cells). For microdissected FFPE tissues, we recommend the RNeasy FFPE Kit. RNeasy technology simplifies total RNA isolation from cells, tissues and yeast by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification. RNeasy Kits provide the highest-quality RNA with minimum copurification of DNA.

Procedure

RNeasy technology simplifies total RNA isolation (see table “Amount of starting material for RNeasy procedures”). Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using a convenient on-column DNase treatment. Pure, concentrated RNA is eluted in water. A variety of special application protocols is also available.

Automated processing on the QIAcube Connect

The QIAcube Connect uses advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into laboratory workflows. All steps in the purification procedure are fully automated — and up to 12 samples can be processed per run. The QIAcube Connect together with the dedicated RNeasy Mini QIAcube Kit provides fast, easy, and convenient RNA purification.

High-throughput processing in 96-well format

The RNeasy 96 system provides a fast and efficient 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 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 the QIAvac 96 vacuum manifold and/or the 96-Well-Plate Centrifuge System.

Amount of starting material for RNeasy procedures

Amount of
starting material
RNeasy Micro Kit RNeasy Mini Kit RNeasy Mini QIAcube Kit RNeasy Midi Kit RNeasy Maxi Kit RNeasy 96 Kit
Cells <5 x 105 cells 10 to 1 x 107 animal or human cells 1 x 107 cells 5 x 106 to 1 x 108 animal or human cells Up to 5 x 108 animal or human cells 10–5 x 105 cells
Tissue <5 mg tissue 0.5–30 mg animal or human tissues 30 mg tissue 20–250 mg animal or human tissues 150 mg to 1 g tissue
Yeast   <5 x 107 yeast cells 2 x 107 to 5 x 108 yeast cells 2.5 x 109 yeast cells

Applications

RNA purified with RNeasy technology has A260/280 ratios of 1.9–2.1 (measured in 10 mM Tris·Cl, pH 7.5) and is ideal for use in all applications. Downstream applications include:

  • RNA-seq
  • Quantitative, real-time RT-PCR
  • Real-time RT-PCR starting with as little as one cell
  • End-point RT-PCR
  • Northern, dot, and slot blotting
  • Array analysis
  • Poly A+ RNA selection

Comparison of RNeasy Kits

Features RNeasy Micro Kit RNeasy Mini Kit RNeasy Mini QIAcube Kit RNeasy Midi Kit RNeasy Maxi Kit RNeasy 96 Kit
Applications RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis
RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis
RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis
RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis
RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis
RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis
Elution volume 10–14 µl 30–100 µl 30–100 µl 300–500 µl 800–2400 µl 45–140 µl
Format Spin column Spin column Spin column, preloaded into rotor adapters Spin column Spin column 96-well plate
Main sample type Tissue, cells Tissue, cells, yeast Tissue, cells, yeast Tissue, cells, yeast Tissue, cells, yeast Cultured cells
Processing Manual (centrifugation) Manual (centrifugation) Automated (QIAcube Connect) Manual (centrifugation) Manual (centrifugation) Manual (centrifugation or vacuum)

Purification of total RNA, miRNA,
poly A+ mRNA, DNA or protein

Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA
Sample amount <5 mg tissue,
<5 x 105 cells
0.5–30 mg tissue,
10 to 1 x 107 animal or human cells
0.5–30 mg tissue,
1 x 107 cells
20–250 mg tissue,
5 x 106 to 1 x 108 animal or human cells
150 mg – 1 g tissue,
Up to 5 x 108 animal or human cells
5 x 105
Technology Silica technology Silica technology Silica technology Silica technology Silica technology Silica technology
Time per run or per prep 30–40 minutes 20 minutes <1 hour 1 hour 30–40 minutes
Yield Varies Varies Varies Varies Varies 1.3–3.1 µg

Supporting data and figures

Resources

Kit Handbooks (5)

For total RNA isolation from animal cells, animal tissues, bacteria, yeast, whole blood, and for RNA cleanup

For RNA isolation from animal and human cells and for RNA cleanup
RNeasy Mini Kit - For purification of total RNA from animal cells, animal tissues, bacteria, and yeast, and for RNA cleanup; RNeasy Protect Mini Kit - For immediate stabilization of RNA in harvested animal tissues and subsequent total RNA purification; RNeasy Plant Mini Kit - For purification of total RNA from plants and filamentous fungi
Quick-Start Protocols (6)
User-Developed Protocols (5)
One milliliter of milk contains approximately 50,000-200,000 leukocytes (with an average of approximately 100,000 leukocytes). In a BVDV-infected animal up to 1-30% of the leukocytes may be infected with the virus. Each infected leukocyte typically contains 10-10,000 BVDV entities.
Supplementary Protocols (7)
Up to 1 x 109 bacteria are disrupted and homogenized by bead-milling in a guanidine-thiocyanate-containing lysis buffer. After addition of ethanol, the sample is loaded onto an RNeasy Mini spin column. Total RNA binds to the RNeasy silica-membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water.
Scientific Posters (1)
Poster for download

Publications

Characterization of immortalized choroid plexus epithelial cell lines for studies of transport processes across the blood-cerebrospinal fluid barrier.
Kläs J; Wolburg H; Terasaki T; Fricker G; Reichel V;
Cerebrospinal Fluid Res; 2010; 7 :11 2010 Aug 12 PMID:20704740
Notch and Wnt signaling mediated rod photoreceptor regeneration by Müller cells in adult mammalian retina.
Del Debbio CB; Balasubramanian S; Parameswaran S; Chaudhuri A; Qiu F; Ahmad I;
PLoS One; 2010; 5 (8):e12425 2010 Aug 26 PMID:20865053
High-definition DNA methylation profiles from breast and ovarian carcinoma cell lines with differing doxorubicin resistance.
Boettcher M; Kischkel F; Hoheisel JD;
PLoS One; 2010; 5 (6):e11002 2010 Jun 8 PMID:20544021

FAQ

Do you have a protocol for purification of cytoplasmic RNA from animal cells?

Yes, please follow the Supplementary Protocol 'Purification of cytoplasmic RNA from animal cells using the RNeasy Mini Kit' (RY25).

 

 

FAQ ID -1257
How can I obtain DNA-free RNA using an RNeasy Midi or Maxi Kit?
The RNeasy Midi/Maxi Handbook contains a protocol for the use of the RNase-Free DNase Set for on-column DNA digestion on RNeasy midi or maxi spin columns. Incubation times and reagent volumes have been modified from the standard RNeasy Mini procedure. See Appendix E of the RNeasy Midi/Maxi Handbook for the detailed protocol.

FAQ ID -143
How much RNA does a typical mammalian cell contain?

The RNA content and RNA make up of a cell depend very much on its developmental stage and the type of cell. To estimate the approximate yield of RNA that can be expected from your starting material, we usually calculate that a typical mammalian cell contains 10–30 pg total RNA.

 

The majority of RNA molecules are tRNAs and rRNAs. mRNA accounts for only 1–5% of the total cellular RNA although the actual amount depends on the cell type and physiological state. Approximately 360,000 mRNA molecules are present in a single mammalian cell, made up of approximately 12,000 different transcripts with a typical length of around 2 kb. Some mRNAs comprise 3% of the mRNA pool whereas others account for less than 0.1%. These rare or low-abundance mRNAs may have a copy number of only 5–15 molecules per cell.

FAQ ID -2946
How is the RNeasy Microarray Tissue Mini Kit different from the classic RNeasy Mini Kit?

The RNeasy Microarray Tissue Mini Kit has been designed for optimal lysis of all types of tissue for subsequent microarray analysis. Lysis and homogenization are performed using QIAzol Lysis Reagent, a phenol-guanidine–based lysis reagent. The aqueous phase is then directly applied to an RNeasy spin column for further RNA cleanup. The RNeasy Microarray Tissue Mini Kit combines optimal lysis of tissues with silica-membrane technology, resulting in high-quality total RNA.

 

 

 

FAQ ID -2192
Which kit should I use for RNA isolation from Cartilage?

Due to the complex nature of cartilage we would recommend to use the RNeasy Lipid Tissue Kit. Follow the standard tissue protocol in the RNeasy Lipid Tissue Kit Handbook.

To help you choose the correct RNeasy kit for the isolation of total RNA from different types of tissue, please refer to our Kit Selection Guide.

FAQ ID -1026
What kits does QIAGEN offer to extract RNA from whole blood?

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
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
What is the smallest sample size that can be used with RNeasy Mini Kits?
We have purified RNA from as little as 0.5 mg tissue or 100 cells using the RNeasy Mini Kit and verified the quality by northern blot analysis and RT-PCR, respectively. In principle, there is no minimum sample amount for RNeasy technology as all RNA will bind to the spin column. However, due to a very small amount of residual RNA irreversibly bound to the silica membrane, not all RNA may elute at the final step of the isolation. The RNeasy Micro Kit can purify RNA from as little as one cell.
FAQ ID -485
Is it possible to use the QuantiTect Reverse Transcription Kit with bacterial RNA?

Yes, it is possible to use the QuantiTect Reverse Transcription Kit for bacteria. The RT Primer Mix provided in the kit is a unique, optimized blend of random primers and oligo-dT allowing high cDNA yields from all regions of RNA transcripts. It has successfully been tested for Reverse Transcription in bacteria as well. We strongly recommend to isolate bacterial RNA using the RNeasy Mini Kit prior to performing Reverse Transcription. This will ensure the high prep quality necessary for optimal RT results with the QuantiTect Reverse Transcription Kit.

FAQ ID -785
I received a kit containing the MinElute columns; however, they were left out for a while and not stored at 2–8°C upon receipt. Can I still use them?

The MinElute spin columns included in the following kits should be stored at 2–8°C upon arrival: AllPrep DNA/RNA Micro, EpiTect Fast DNA Bisulfite, EpiTect Fast FFPE Bisulfite, EpiTect Fast LyseAll Bisulfite, EpiTect Plus DNA Bisulfite, EpiTect Plus FFPE Bisulfite, EpiTect Plus LyseAll Bisulfite, exoRNeasy Serum/plasma Maxi, exoRNeasy Serum/Plasma Midi, GeneRead DNA FFPE, GeneRead rRNA Depletion, GeneRead Size Selection, MinElute Gel Extraction, MinElute PCR Purification, MinElute Reaction Cleanup, miRNeasy FFPE, miRNeasy Micro, miRNeasy Serum/Plasma, QIAamp DNA FFPE, QIAamp DNA Investigator, QIAamp DNA Micro, QIAamp MinElute Media, QIAamp MinElute Virus Spin, QIAamp MinElute Virus Vacuum, RNeasy FFPE, RNeasy Micro, RNeasy Plus Micro.

Short-term storage (up to 4 weeks) at room temperature (15–25°C) does not affect the performance. However, for optimal performance and quality, storage temperature should not exceed 25°C.

FAQ ID - 3560
Is it possible to isolate both RNA and recombinant 6xHis-tagged protein from the same sample?
We have no experimental data for this application. However, buffer RLT of the RNeasy Kits for RNA isolation does not contain substances incompatible with Ni-NTA purification of His-tagged proteins. It should be possible to first extract RNA from a sample by following the RNeasy procedure, save the flow-through from the binding step as well as from the RW1 wash, and apply the combined fractions onto a Ni-NTA column for binding of His-tagged proteins. Follow our recommendations for purification of 6xHis-tagged proteins using Ni-NTA resins outlined in the QIAexpressionist handbook.
FAQ ID -532
Is RNAprotect Bacteria Reagent compatible with the RNeasy Mini Kit?

Yes, RNAprotect Bacteria Reagent and the RNeasy Mini Kit are compatible. You can also purchase QIAGEN's RNeasy Protect Bacteria Kits, which will contain RNAprotect Bacteria Reagent and RNeasy chemistry. Protocols for isolating RNA from bacteria after using the Reagent for bacterial RNA stabilization can be found in the RNAprotect Bacteria Reagent Handbook.

FAQ ID -797
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 avoid little or no RNA yields when using an RNeasy Kit?

Avoid RNA degradation due to improper sample storage and handling prior to the extraction procedure with RNeasy Kits. RNA in tissues is not protected after harvesting until the sample is treated with RNAprotect Tissue Reagent, flash frozen, or disrupted and homogenized in the presence of RNase-inhibiting or denaturing reagents. Samples can be immediately flash frozen in liquid nitrogen and stored at −90 to −65°C. as soon as they are harvested or excised. Frozen tissue should not be allowed to thaw during handling or weighing, but cell pellets can partially thaw enough to allow them to be dislodged by flicking. The relevant procedures should be carried out as quickly as possible. Samples can also be stored at −90 to −65°C. in lysis buffer (Buffer RLT) after disruption and homogenization. Frozen samples are stable for months.

For optimal RNA yields with RNeasy Kits it is crucial to:

  • Efficiently disrupt and homogenize the starting material.
  • Use the correct amount of starting material (do not overload!).
  • Perform all protocol steps at room temperature.
  • Perform the dry-spin prior to elution as described in the relevant protocol for a full 5 minutes.
  • Prepare the 80% ethanol for the wash steps with RNase-free water only.
  • Dispense the RNase-free water for elution onto the center of the membrane.
  • Optionally, repeat the elution step, and incubate the spin column on the bench for 10 minutes with RNase-free water before centrifuging.

Please review the instructions in the relevant RNeasy Handbook carefully for best results.


FAQ ID -28
What is the minimum elution volume when using the RNeasy Micro Kit?
The minimum elution volume for the RNeasy MinElute Spin Columns used in the RNeasy Micro Kit is 10 ul.
FAQ ID -428
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
Why is it not recommended to stabilize cells with RNAprotect Tissue Reagent?

RNAprotect Tissue Reagent is recommended for stabilization and protection of RNA in animal tissues. It is not recommended for cultured cells because it may be difficult to pellet and remove them from the reagent prior to RNA isolation (see FAQ 364). 

Instead, we recommend to use the RNAprotect Cell Reagent.  This reagent is for immediate RNA stabilization in sorted or cultured cells with no need to remove medium

 

FAQ ID -941
Can the RNeasy kits be used to extract RNA from saliva collected using the Oragene collection kit?

There is a protocol available at the manufacturers (DNA Genotek) homepage: http://www.dnagenotek.com/US/pdf/PD-PR-021.pdf

There is also a White Paper available demonstrating the performance: http://www.dnagenotek.com/US/pdf/PD-WP-008.pdf

FAQ ID - 3623
What is the maximum binding capacity of RNeasy spin columns?
The maximum binding capacity of the RNeasy Mini spin columns is 100 ug RNA. It is 1 mg for RNeasy Midi columns and 6 mg for RNeasy Maxi columns. The maximum RNA binding capacity of the RNeasy MinElute spin columns is 45 µg.
FAQ ID -290
Do you have a protocol for isolating cytoplasmic RNA from animal cells using the RNeasy Mini Kit?

Yes, please follow the Supplementary Protocol 'Purification of cytoplasmic RNA from animal cells using the RNeasy® Mini Kit' (RY25).

 

FAQ ID -1207
What is the composition of PBS?

The composition of PBS is:

  • 50 mM potassium phosphate
  • 150 mM NaCl; pH 7.2

PBS solution is not a component of any QIAGEN kit, but is used in protocols for recombinant protein purification with the QIAexpress System, and in some protocols for DNA and RNA isolation with various QIAGEN Kits.

 

FAQ ID -361
Do you have a protocol for the isolation of RNA from leukocytes in milk?
Yes, please follow the User-Developed Protocol 'Isolation of RNA from leukocytes in milk using QIAGEN RNeasy Kits' (RY12).
FAQ ID -952
What is the key technical challenge in isolating high quality RNA from cell or tissue samples?

Ribonucleases are the principal threat to any RNA isolation procedure. In addition, copurification of inhibitory contaminants is a major problem when isolating RNA from certain tissue sources. To minimize the threat, gloves should be worn at all times, and special care must be taken to use RNase-free reagents and labware.

In addition, tissue/cell lysis steps are typically carried out with lysis buffers containing guanidine isothiocyanate, a potent protein denaturant. It is very important to use a sufficient amount of lysis buffer during RNA isolation. We recommend using at least 10X volume of lysis buffer to tissue/cell pellet.

In general, for fast purification of high-quality RNA we recommend QIAGEN’s RNeasy Kits. It is more challenging to isolate high-quality RNA from tissue samples than from cultured cells, especially those tissues containing high levels of RNase, or difficult-to-homogenize tissues. Examples of such tissues include liver, heart, skin, and conjunctive tissues. Many tissue samples also contain difficult-to-remove contaminants (such as polysaccharides, collagen, fats, lipids or fibrous components) that may interfere with subsequent enzymatic reactions if not removed from the RNA preparation. For purification of high-quality RNA from difficult tissues we recommend QIAGEN’s RNeasy Plus Universal Kit.

FAQ ID -2656
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
Can QIAquick Kits be used to clean up RNA samples?
Although it is possible to use the QIAquick Nucleotide Removal Kit and QIAquick PCR Purification Kit for RNA purification, the conditions are not optimized for RNA, nor are RNase-free conditions guaranteed. The RNeasy MinElute Cleanup Kit is recommended for clean-up, concentration and desalting of RNAs above 200 bases in length.
FAQ ID -490
Is it possible to perform laser microdissection with tissues stabilized with RNAprotect Tissue reagent?

For Laser Captured Microdissection, its best to either flash-freeze the tissue and do cryosectioning, or to use FFPE material. 

FAQ ID -608
What is a QIAshredder? Is it sufficient for complete disruption and homogenization of my tissue sample?

The QIAshredder is a unique biopolymer shredding system in a microcentrifuge spin-column format. It homogenizes cell or tissue lysates to reduce viscosity. Homogenization shears the high-molecular weight genomic DNA and other high-molecular-weight cellular components to create a homogenous lysate. The QIAshredder is chemically inert and will not bind nucleic acids.

It cannot replace tissue disruption or enzymatic cell wall lysis by mechanical and chemical methods, respectively. Both, efficient cell wall disruption and lysate homogenization are fundamental for successful RNA isolation from all types of samples. Once complete disruption has been achieved, the QIAshredder Homogenizer can be used in place of needle and syringe, or rotor-stator homogenization.

For further information regarding sample disruption and homogenization please refer to the 'Disruption and homogenization of starting materials' section in the RNeasy Mini Handbook, and see FAQ 139.

 

FAQ ID -631
Do you have a protocol for the isolation of total RNA from buccal swabs?
FAQ ID -956
What is the binding capacity of the RNeasy Micro Kit column?
The maximun binding capacity of the RNeasy MinElute Spin column supplied in the RNeasy Micro Kit is 45 ug of RNA.
FAQ ID -427
Are RNeasy spin columns sold separately?
At this time, RNeasy spin columns are not sold separately.
FAQ ID -159
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 are the most reliable methods for preparing high-quality RNA from cell or tissue samples, for use in gene expression analysis experiments?
We recommend the use of RNeasy Mini Kits. Cultured cells, and freshly isolated white blood cells, may be harvested by centrifugation, and used directly with this kit. For the isolation of high-quality RNA from animal tissues, we recommend RNeasy Plus Universal Kit.
FAQ ID -2657
What is the composition of Buffer RDD?
The exact composition of Buffer RDD is proprietary. Buffer RDD is an important component of the RNase-Free DNase Set, which is used in combination with most RNeasy Kits. The composition and salt concentration of Buffer RDD provides efficient on-column digestion of DNA and also ensures that the RNA remains bound to the column.
FAQ ID -2800
What is the maximum volume of RNA in solution that can be used with the QuantiTect Whole Transcriptome Kit?

A maximum of 5 µl RNA eluate from RNeasy extraction procedures can be added to the reverse-transcription reaction with the QuantiTect Whole Transcriptome Kit.

 

 

FAQ ID -1616
How can I improve my RNA yield from liver sample when using RNeasy Kits?
Sometimes, liver causes difficulties when used as starting material for the RNeasy Mini/Midi/Maxi Protocol for isolation of total RNA from Animal Tissue. This can be resolved by adding 1 volume of 50% ethanol to the cleared RLT lysate before applying it to the RNeasy column, instead of the 70% ethanol specified in the RNeasy Mini Kit Handbook and RNeasy Midi/Maxi Kit Handbook.
FAQ ID -623
Can I clean up my DNase treated RNA samples using RNeasy columns?
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
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
What is the minimum number of cells or minimum amount of tissue that can be efficiently processed with the RNeasy mini kit?
In QIAGEN labs we have successfully isolated RNA from a minimum of 100 HeLa or Jurkat cells without carrier RNA. The Lowest amount of tissue we have used is 0.5 mg. The quality and quantity was verified with RT-PCR. When added to lysates from very small samples, the carrier RNA may in some cases improve the recovery of total RNA. For 30µl of lysate, add 20 ng of carrier RNA. Carrier RNA is not required when processing more than 500 cells or more than about 2 μg tissue. However, please bear in mind that for these small quantities, we would recommend the RNeasy Plus Micro Kit instead.
FAQ ID - 3519
How is the RNeasy Lipid Tissue Mini Kit different from the RNeasy Mini Kit?
The RNeasy Lipid Tissue Kits have been designed for the optimal lysis of tissues rich in fat, such as brain and adipose tissues. Lysis and homogenization is performed using QIAzol, a phenol/guanidine-based lysis reagent. The RNA is then purified from the aqueous fraction using a RNeasy spin column. The RNeasy Lipid Tissue Kit combines optimal lysis of fatty tissues with silica membrane technology, resulting in high-quality total RNA.
FAQ ID -468
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
What do you suggest to prevent degradation of RNA isolated from tissue with high amounts of RNases using RNeasy?

RNA can efficiently be stabilized in tissue samples by using RNAprotect Tissue Reagent. If the sample is frozen, it should not be allowed to thaw prior to disruption and homogenization in Buffer RLT or QIAzol lysis reagent of the RNeasy Kits.

In addition, you can increase the percentage of beta-Mercaptoethanol added to Buffer RLT from 1% (v/v) to 3-5% (v/v) to enhance denaturation and inactivation of RNases.


FAQ ID -942
How should I reconstitute the lyophilized RNase-Free DNase Set with the RNeasy 96 kit?

RNeasy 96 Kit procedure requires two RNase-Fee DNase sets (cat. no. 79254) for each 96-well plate.

 

1. To obtain a concentration of 1.8 Kunitz/µl, dissolve each vial of lyophilized DNase in 833 µl of RNase-free water and combine the reconstituted enzyme solution from both vials into one vial.

2. Take 1 ml of this DNase I solution and mix with 7 ml of Buffer RDD* to prepare a DNase I incubation mix immediately before starting the RNeasy 96 protocol. Vortex briefly and keep on ice until use.

3. Use 80 µl of the DNase I incubation mix (from step 2 above) for each well of the RNeasy 96 plate, to perform on-column DNase digestion, according to instructions in the RNeasy 96 Handbook.

 

*The RDD Buffer Set for RNeasy 96 is available upon request.  Please contact QIAGEN Technical Service for information on ordering.  Buffer RDD from QIAGEN is optimized for DNase I digestion on the RNeasy membrane.

FAQ ID -3003
What is the difference between Buffers RLT and RLT Plus?

In comparison to Buffer RLT of, e.g., the RNeasy Mini Kit, Buffer RLT Plus of the RNeasy Plus Mini Kit and RNeasy Plus 96 Kit also contains a proprietary blend of detergents that aid in the binding of genomic DNA to the gDNA Eliminator Mini Spin Columns, or to the gDNA Eliminator 96 plate respectively.

FAQ ID -1043
Can acetone be used for precipitation of protein from Buffer RLT lysates generated with RNeasy Kits?

Yes, please follow the Supplementary Protocol Acetone precipitation of protein from Buffer RLT or Buffer RLT Plus lysates (RY22).

Important Note:

Do not use TCA to precipitate protein from Buffer RLT and Buffer RLT Plus lysates. These buffers contain guanidine thiocyanate, which can form highly reactive compounds when combined with acidic solutions.

 

For simultaneous purification of DNA, RNA, and protein from the same sample (either cultured cells or easy-to-lyse tissues), we recommend using the AllPrep DNA/RNA/Protein Mini Kit. This kit allows precipitation of protein from Buffer RLT lysates using a novel protein precipitation buffer, Buffer APP.

Please note that Buffer APP is not compatible with Buffer RLT Plus. Acetone should be used instead to precipitate protein from RLT Plus lysates.

 

FAQ ID -1164
Do you have a protocol for the isolation of total RNA from plants using the RNeasy 96 Kit?
Yes, please follow the User-Developed Protocol 'Isolation of total RNA from plants using the RNeasy 96 Kit' (RY23).
FAQ ID -951
What do you recommend for isolating RNA from more than 100 mg of plant tissue?
You can use the RNeasy Midi Kit for up to 500 mg of plant tissue, and the RNeasy Maxi Kit for up to 1 g of plant material. Please follow the guidelines in Appendix D of the RNeasy Midi/Maxi Handbook. The samples will require rotor-stator homogenization, since QIAshredders are not available for the RNeasy Midi/Maxi format.
FAQ ID -570
How do I clean up RNA preparations containing miRNA?

RNA preparations containing miRNA can be cleaned up by modifying* the cleanup protocols listed in the handbooks of the RNeasy Mini Kit or the RNeasy MinElute Cleanup Kit .

 

* Modify the cleanup protocol at step 2, by increasing the volume of ethanol (96-100%) from 250 µl to 950 µl.

FAQ ID -3002
How much time can be saved when using the RNeasy Mini QIAcube Kit?
Processing your samples with the QIAcube saves 80% hands-on time compared to the manual procedure. Using the RNeasy Mini QIAcube Kit saves another 10% hands-on time compared to processing the classic RNeasy Mini Kit on the QIAcube.
FAQ ID -2335
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
Do I have to discard Buffer RLT with beta-Mercaptoethanol (ß-ME) added to it after 1 month of storage?
No. Beta-Mercaptoethanol (ß-ME) is stable for 1 month, but Buffer RLT itself is stable for at least 9 months at room temperature (15 to 25°C). Simply add fresh ß-ME to the Buffer RLT supplied in RNeasy Kits to ensure complete inactivation of RNases while isolating RNA.
FAQ ID -1037
Which kit should be used to extract RNA from adipose tissue, brain, and other fatty animal tissues?
The RNeasy Lipid Tissue Mini and Midi Kits are designed to extract RNA from up to 100mg and 500mg of fatty tissue, respectively.
FAQ ID -467
Are the buffers in the RNeasy QIAcube kit delivered in QIAcube buffer bottles?
No, in order to reduce waste as far as possible the buffers of the RNeasy Mini QIAcube Kit are not delivered in QIAcube buffer bottles but in bigger dimensioned standard QIAGEN buffer bottles. The buffers still have to be transfered into QIAcube buffer bottles by hand.
FAQ ID -2334
How can I isolate RNA from 1 gram of plant tissue?
For large-scale RNA isolation from plants, 500 mg of tissue can be processed using the RNeasy Midi Kit, and 1 g using the RNeasy Maxi Kit. With optimization, it may be possible to use even larger amounts of starting material. Please refer to Appendix D of the RNeasy Midi/Maxi Handbook for guidelines for plant Midi- and Maxi preps.

FAQ ID -128
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 efficient is DNA removal by a gDNA Eliminator 96 plate compared with DNase digestion on an RNeasy 96 plate?

Both DNase digestion on the RNeasy 96 plate as well as the gDNA Eliminator 96 plate with RNeasy Plus 96, eliminate DNA contamination with similar efficiency.  However, using the gDNA Eliminator 96 plate of the RNeasy Plus 96 Kit is faster and more economical. 

 

 

FAQ ID -1994
3326 - Which QIAcube standard protocol might be suitable to extract RNA from saliva or from a buccal cell pellet?
The RNeasy Micro protocol with a DNase Digestion step has the same workflow as the manual RNeasy Protect Saliva Mini Kit, which is designed for saliva samples.
Can the QIAshredder Maxi Spin Columns from the DNeasy Plant Maxi Kit be used for RNeasy samples?

The QIAshredder Maxi spin columns  from the DNeasy Plant Maxi Kit are not recommended for use in conjunction with QIAGEN's RNA isolation products. The filter can only tolerate low centrifugal forces not sufficient to shear genomic DNA. If genomic DNA is not sheared, there will be an increase in genomic DNA contamination in the purified RNA.

For an alternative to QIAshredder homogenization in combination with RNeasy Midi/Maxi Preps, please see FAQ 560.

FAQ ID -616
What is the maximum amount of starting material that can be processed with the RNeasy Micro Kit?
The RNeasy Micro Kit can be used for the isolation of RNA from up to 5x105 cells, or up to 5 mg of tissue. It can also be used for the cleanup of up to 45 ug of RNA in solution.
FAQ ID -426
How many samples can be processed per RNeasy Mini QIAcube kit?
The RNeasy Mini QIAcube Kit contains preloaded rotor adapters for 240 samples or 20*12 runs on the QIAcube.
FAQ ID -2332
Do you offer QIAshredder spin columns for the RNeasy Midi/Maxi Kit format? If not, what is the alternative?
We only offer QIAshredder spin columns for use with the RNeasy Mini Kit. When working with RNeasy Midi/Maxi, we recommend using a rotor-stator, or mortar and pestle with needle and syringe homogenization.
FAQ ID -560
What dedicated QIAcube Kits are available?
Will the "classic" RNeasy Mini Kit be discontinued after the launch of the Allprep DNA/RNA Mini Kit?

No, our standard RNeasy Mini Kit will not be discontinued. The Allprep DNA/RNA Mini Kit is an addition to our RNeasy product line.

 

 

FAQ ID -1054
How can I minimize degradation of RNA from my pancreas sample?

Pancreas is very high in RNases. Therefore, it is important to minimize the time between harvesting the tissue and snap freezing or stabilization in RNAprotect Tissue Reagent.  Use of 3-5% ß-mercaptoethanol in Buffer RLT instead of 1% may also improve the results.


FAQ ID -624
What is Tissue-Tek O.C.T., and what is it used for?

Tissue-Tek O.C.T. is an embedding compound for cryosectioning, which is soluble in water. It mainly consists of glycols and synthetic resins. Tissue-Tek O.C.T. is used as matrix for cryosectioning of tissues. Using the O.C.T. the tissue samples can be positioned more easily in the microtome and have better qualities during sectioning.

Most cryosections are fixed using non-crosslinking agents. For isolation of RNA from tissue embedded in Tissue-Tek O.C.T. using non-crosslinking agents the RNeasy Plus Micro Kit or the RNeasy Micro Kit (Protocol: Total RNA Isolation from Microdissected Cryosections), or the RNeasy Mini Kit (RNeasy Mini Protocol for the isolation of Total RNA from Animal Tissue) give great results. We strongly recommend removing as much of the embedding compound as possible prior to RNA extraction from the sections. Please see QIAGEN News article, Issue 1 1998, 'Effects of malnutrition on expression of lactase in children' for successful RNA isolation from O.C.T.-embedded tissue using the RNeasy Mini Kit.

In case crosslinking agents (e.g. formaldehyde or glyoxal-containing) were used for fixation of the tissue for cryosectioning the RNeasy FFPE Kit is the perfect choice. The RNeasy FFPE Kit is especially designed for purifying total RNA from formalin-fixed tissue sections. Special lysis and incubation conditions reverse formaldehyde modification of RNA for improved results in downstream application. The crosslinking causes the RNA to break, resulting in overall smaller molecules, which look like a smear when analyzed on a formaldehyde gel.

In case DNA as well as RNA from precious samples is of interest from the identical sections, please have a look at the AllPrep DNA/RNA Kits for unfixed tissue or the AllPrep DNA/RNA FFPE Kit for fixed tissue.

FAQ ID -530
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
Do I need to use RNase inhibitors with the RNeasy Kits?

No. The addition of beta-mercaptoethanol to lysis buffer RLT used in the RNeasy Kits is sufficient to inactivate any RNases in your sample. 

FAQ ID -813
Have you observed co-amplification of genomic DNA from RNA templates used in the QuantiTect Whole Transcriptome Procedure?

No, we have never observed coamplification of genomic DNA from RNA templates used in the QuantiTect Whole Transcriptome protocol when using RNA purified with RNeasy Kits without on-column DNase digestion.

 

 

FAQ ID -1619
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
Are QIAshredder spin columns delivered with the RNeasy Mini QIAcube Kit?
No, QIAshredder spin columns are not included in the RNeasy Mini QIAcube Kit but can be ordered separately.
FAQ ID -2333
I accidentally added RNAprotect Tissue Reagent to my cells, and now the cells are difficult to pellet. What can I do?

If the cells in RNAprotect Tissue Reagent cannot be collected by centrifugation, please try one of the following suggestions:

1) Add 600 µl Buffer RLT to a maximum of 200 µl sample volume, and proceed with step 3 of the "RNeasy Mini Protocol for Isolation of Total RNA from Animal Cells" in the 
RNeasy Mini Handbook. Load the lysate onto the column in successive aliquots in step 5 of the protocol.

2) If cells are floating on the surface of the RNAprotect Tissue Reagent, try removing the reagent by pipetting from underneath. Leave behind approximately 100 ul of RNAprotect Tissue Reagent, and add 350 ul Buffer RLT before proceeding with the protocol "RNeasy Mini Protocol for Isolation of Total RNA from Animal Cells". For every 100 ul of cells in RNAprotect Tissue Reagent, use 250 ul of 96-100% ethanol instead of the 70% ethanol listed in step 4 of the standard protocol.

3) Dilute the sample 10x by adding cold PBS. Pellet cells by centrifugation. Caution: Cells might lyse.

4) Try to pipette the floating cells off the surface.

Note that the above steps are suggestions, rather than official protocol recommendations. Please try a "pilot" run on a test sample first.

For RNA stabilization of cells, we recommend using the RNAprotect Cell Reagent.

 

FAQ ID -364
How can I ensure complete genomic DNA removal when using the RNase-Free DNase Set?

To ensure efficient gDNA removal when doing an on-column digest using the RNase-Free DNase Set in combination with RNeasy Mini the following factors are crucial:

  • prevent overloading by adjusting the amount of starting material to no more than the maximum amounts recommended in the RNeasy Mini Handbook
  • ensure complete disruption and homogenization of the starting material as instructed in the section 'Disruption and homogenization of starting materials' of the handbook
  • strictly follow the protocol for on-column DNase Digestion in Appendix D of the RNeasy Mini Handbook (you can let wash buffer RW1 incubate on the column for 3-5 minutes before centrifuging to enhance removal of excess gDNA prior to applying the enzyme)

In the rare case that trace amounts of genomic DNA are still detectable in sensitive downstream applications such as e.g., realtime RT-PCR, an in-solution digest using the RNase-Free DNAase set can be performed. Instructions are presented in Appendix C of the RNeasy MinElute Cleanup Handbook.

Alternatively, a second on-column digest can be carried out in future preparations, immediately following the RW1 wash after the first incubation with DNase.

FAQ ID -1087
What happens if I spin my lysate on the RNeasy Spin Columns at maximum speed?
Spinning at maximum speed is fine, since binding of RNA to the columns will also be efficient. Instead, the critical issue is not to spin the columns below the minimum speeds recommended in the RNeasy Handbooks.
FAQ ID -514
Why does my isolated RNA have a low OD 260/280 ratio?

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
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
Why do I have to add beta-mercaptoethanol (beta-ME) to lysis Buffer RLT of the RNeasy Kits?

When working with RNA, care must be taken to avoid degradation by RNases, which are extremely stable and active. Intracellular RNases are released during the lysis step of the RNA isolation procedure and must be rapidly and thoroughly inactivated to obtain high-quality RNA.

Beta-mercaptoethanol (ß-ME) is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for enzyme functionality. In combination with the strong, but temporary denaturing effects of guanidinium isothiocyanate (GITC) contained in buffer RLT of the RNeasy Kits, any RNases present in the material to be extracted from will be completely inactivated.

FAQ ID -101
What sample types can be used with the AllPrep DNA/RNA 96 Kit?

The Allprep DNA/RNA 96 kit is designed for cultured human or animal cells, and for easy-to-lyse human or animal tissues. Tissues compatible with RNeasy Mini, RNeasy Plus Mini, and RNeasy Plus 96 are also compatible with the Allprep DNA/RNA 96 kit.

For more information on compatible kits and sample types, see our Selection Guide for RNA purification.

FAQ ID -1996
What should I do if I suspect that my RNA preparation contains RNase contamination?

If you suspect that your RNA preparation contains RNase contamination, repurify the preparation using a RNeasy Mini Kit or RNeasy MinElute Cleanup Kit.

.

FAQ ID -2661
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
Do you have a protocol for isolating RNA from yeast using RNeasy?

Yes, the RNeasy Mini Kit, RNeasy Midi Kit and RNeasy Maxi Kit can be used for this purpose. The RNeasy Mini Kit Handbook contains a standard and an abbreviated protocol using enzymatic lysis, and one protocol using mechanical disruption. The RNeasy Midi/Maxi Handbook contains a standard protocol for enzymatic lysis and one for mechanical disruption. The enzymatic protocols employ zymolase and lyticase, and the mechanical disruption protocols employ glass beads and a beadmill for cell wall disruption.

FAQ ID -535
Can I use the RNeasy Mini Kit or RNeasy 96 Kit with fewer than 100 cells?

Yes. The RNeasy Mini Kit and RNeasy 96 Kit have been used successfully to isolate RNA from fewer than 100 cells. We recommend adding 20 ng of carrier RNA to the cell lysate before loading it onto the RNeasy membrane. Please note that the carrier RNA will co-purify with cellular RNA. However, the small amounts of poly-A RNA used as carrier RNA do not interfere with subsequent RT-PCR, even when oligo-dT is used for reverse transcription. Reverse-transcription reactions typically contain a large excess of oligo-dT, and the small amounts of poly-A used as carrier RNA are insignificant in comparison.

Alternatively, the RNeasy Micro Kit can be used for isolating RNA from up to 5x 105 cells. The RNeasy Micro procedure uses a novel technology to purify RNA from small amounts of tissues or cells (as little as 1 cell).

FAQ ID -372
Can the QuantiTect Whole Transcriptome Kit be used for amplification of RNA from LCM samples?

Yes, use the RNeasy Micro Kit or RNeasy Mini Kit to purify RNA from laser-capture microdissected (LCM) samples prior to amplification with the QuantiTect Whole Transcriptome Kit. Note, however, that carrier RNA has to be avoided in the RNA purification procedure as it may affect specific amplification of transcript sequences.

RNeasy Kits ensure that the RNA is of sufficient quality. Degraded RNA cannot be used for amplification.

 

FAQ ID -1612
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