RNeasy Kits for RNA Purification

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.

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.

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.

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.

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.

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.

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).

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.

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

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

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

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

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.).

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. 

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.

Dedicated QIAcube kits are the RNeasy Mini QIAcube Kit, QIAamp DNA Mini QIAcube Kit, the QIAamp DNA Blood Mini QIAcube Kit, and the QIAamp Viral RNA Mini QIAcube Kit. In addition to these kits are the QIAamp PowerFecal Pro DNA QIAcube Kit, the DNeasy PowerSoil Pro QIAcube Kit using Power technology, and the DNeasy Blood & Tissue QIAcube 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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

.

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.

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.

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).

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.