RNeasy Protect Kits – RNA Stabilization and Isolation

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

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.

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.

Yes, RNAprotect Cell Reagent of the RNeasy Protect Cell Mini Kit can be purchased separately in a bulk size of 250 ml for processing large cell culture volumes.

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.

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.

We do not recommend to store previously isolated RNA in RNAprotect Tissue Reagent, since it will be very difficult to recover isolated RNA from it. Purified RNA can be stored at −30 to −15°C or −90 to −65°C in sterile RNase-free water. Under these conditions, we found no detectable RNA degradation after 1 year of storage.

No, RNAprotect Saliva Reagent is provided in the RNeasy Protect Saliva Mini Kit only, and is not available separately.

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.).

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.

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. 

Yes, but you have to make sure that the RNAprotect Bacteria Reagent is diluted in PBS or water prior to use (2 volumes reagent : 1 volume PBS or water) in order to achieve its optimal working concentration. The diluted reagent is directly added to the bacteria growing on the solid substrate, and reagent and bacteria are mixed thoroughly. The reagent is not recommended for stab cultures, since sufficient access of the reagent to all bacteria for immediate RNA stabilization cannot be guaranteed.

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.

Tissues preserved in RNAprotect Tissue Reagent can be stored as long as listed below:

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.

The RNeasy Protect Saliva Mini Kit is based on RNeasy MinElute technology, which allows elution volumes as low as 10-14 µl, thereby concentrating saliva RNA.

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 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 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).