RNeasy Plus Kits for RNA Isolation

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).

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

During the gDNA elimination step of the RNeasy Plus 96 procedure, the flow-through of the gDNA Eliminator 96 plate has to be collected. If the plate were to be processed in a vacuum manifold, there would be a substantial risk of cross-contamination due to foaming of the lysate. Also, plate drying and RNA elution are more efficient with a centrifuge than with a vacuum manifold.

The RNeasy Plus 96 Kit allows extraction of RNA from up to 1 x 10e6 cells with the vacuum/spin protocol, or up to 2 x 10e6 cells with the spin protocol.

The RNeasy Plus Universal Mini Kit has been designed for optimal lysis of all types of tissue. Lysis and homogenization are performed using QIAzol Lysis Reagent, a phenol-guanidine based lysis reagent. The RNeasy Plus Mini Kit is the optimal solution for cells and easy-to-lyse tissue since lysis occurs with Buffer RLT. Additionally, the gDNA removal feature is based on a different technology. While the classic RNeasy Plus Kit contains gDNA eliminator columns, the RNeasy Plus Universal Kit contains a gDNA eliminator solution.

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.

No. Tissue lysis generates more debris than cell lysis. This additional debris is not compatible with the special binding conditions for small RNAs using the RNeasy Plus 96 Kit, and can lead to a reduction of up to 50% or more in RNA yield.

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.

When using the RNeasy Plus Micro and RNeasy Plus Mini Kits, or the Allprep DNA/RNA Mini Kit, foaming of lysates can be substantially reduced by adding Reagent DX to Buffer RLT Plus at a final concentration of 0.5% (v/v) before starting sample lysis.

Reagent DX has been carefully tested with RNeasy Plus and Allprep Kits and has no effect on RNA purity or on downstream applications. Reagent DX can be purchased separately using catalog number 19088.

For more information on efficient sample disruption and homogenization for nucleic acid extractions, please see further product details and resources for the TissueRuptor, TissueLyser LT, and TissueLyser II.

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. 

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.

In our labs, we were able to purify RNA using the RNeasy Plus Micro Kit, or DNA/RNA using the AllPrep DNA/RNA Micro Kit at single cell level. However, generally we do not recommend purification from fewer than 10–20 cells, due to stochastics related to transcript copy numbers present in a single cell.

For example, if an RNA transcript is present at only 1 copy per cell, recovery from a purification using e.g., 10 cells or less, would be less than 1 transcript copy per microliter, given the recommended elution volume of 14 µl. 

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.

No, Buffer APP for protein precipitation in the Allprep DNA/RNA/protein Mini Kit is not compatible with Buffer RLT Plus. Acetone can be used to precipitate proteins from samples homogenized in Buffer RLT Plus contained in the Allprep DNA/RNA Mini Kit and RNeasy Plus Mini Kit.  

Yes, RNAs smaller than 200 nucleotides, including miRNA, can be isolated using the AllPrep DNA/RNA Micro or RNeasy Plus Micro Kit. Please follow the specialized protocol 'Purification of total RNA containing small RNAs from cells' in Appendix D of the AllPrep DNA/RNA Micro or the RNeasy Plus Micro Handbook, respectively.

Please note that this protocol is for use with cultured cells only, and is not compatible with tissues.

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 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.

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.

We do not sell the gDNA eliminator columns separately.