RNeasy Plus Micro Kit

For purification of up to 45 µg total RNA from cells/tissues using gDNA Eliminator columns

Features

Unique gDNA Eliminator columns avoid the need for DNase
Efficient removal of genomic DNA
Highly reproducible yields of RNA in minutes
High-performance RNA for sensitive applications

Useful Resources

RNeasy Plus Micro Handbook Acetone precipitation of protein from Buffer RLT or Buffer RLT Plus lysates - (EN) RNeasy Plus Micro Kit (EN)

RNeasy Plus Micro Kit (50)

Cat. No. / ID: 74034

For 50 micropreps: RNeasy MinElute Spin Columns, gDNA Eliminator Spin Columns, Collection Tubes, Carrier RNA, RNase-Free Water and Buffers

The RNeasy Plus Micro Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The RNeasy Plus Micro Kit integrates fast, convenient purification of up to 45 µg RNA with effective elimination of genomic DNA. Cell or tissue lysates are spun through gDNA Eliminator spin columns to remove genomic DNA. Total RNA is then purified using RNeasy MinElute spin columns. Tissue samples can be conveniently stabilized using RNAprotect Tissue Reagent or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor or TissueLyser system. For larger samples, the RNeasy Plus Mini Kit (spin-column binding capacity of 100 µg RNA) is also available. Procedures can be fully automated on the QIAcube Connect.

Performance

The RNeasy Plus Micro procedure integrates selective removal of double-stranded DNA with RNeasy technology. After genomic DNA removal, high-quality RNA is purified using RNeasy MinElute spin columns. This allows high, reproducible RNA yields and effective elimination of genomic DNA contamination for sensitive applications (see figure "High, reproducible RNA yields"). Total RNA with Agilent RIN values of close to 10 is routinely obtained from tissues and cultured cells.

Principle

Cells and easy-to-lyse tissues are first lysed and homogenized in highly denaturing guanidine-isothiocyanate–containing Buffer RLT Plus, which immediately inactivates RNases to ensure isolation of intact RNA. The lysate is then passed through a gDNA Eliminator spin column that, in combination with the high-salt buffer, selectively and efficiently removes genomic DNA. Ethanol is added to provide appropriate binding conditions for RNA, and the sample is applied to an RNeasy MinElute spin column. These specialized columns each contain a silica membrane that specifically binds RNA from lysed cells.

Procedure

Total RNA is purified from up to 5 x 10⁵ cells or 5 mg tissue, making the RNeasy Plus Micro Kit well suited for small samples. A short workflow enables RNA purification with genomic DNA removal in less than 30 minutes (see flowchart "RNeasy Plus procedure"). Samples are first lysed and homogenized. The lysate is passed through a gDNA Eliminator spin column, ethanol is added to the flow-through, and the sample is applied to an RNeasy MinElute spin column. RNA binds to the membrane and contaminants are washed away. High quality RNA is eluted in as little as 14 µl water.

Different protocols are available for different starting materials. The protocols differ mainly in the lysis and homogenization of the sample. Once the sample is applied to the gDNA Eliminator spin column, the protocols are similar. The procedure provides an enrichment for mRNA since most RNAs <200 nucleotides (such as rRNAs and tRNAs) are excluded. The RNeasy Plus procedure can be modified to allow the purification of total RNA containing small RNAs, such as miRNA, from cultured cells.

When disrupting and homogenizing tissues in Buffer RLT Plus (supplied with the RNeasy Plus Micro Kit), excessive foaming may occur. This foaming is substantially reduced by adding Reagent DX (supplied separately) to Buffer RLT Plus before starting disruption and homogenization. Reagent DX has been carefully tested with the kit, and has no effect on RNA purity or on downstream applications.

Applications

RNA purified using the RNeasy Plus Micro Kit is ideally suited for downstream applications that are sensitive to low amounts of DNA contamination, such as quantitative real-time RT-PCR. The purified RNA can also be used in other applications.

The RNeasy Plus Micro Kit is suitable for small samples, including:

Laser-microdissected cryosections
Fine-needle aspirates

Supporting data and figures

High, reproducible RNA yields.

Total RNA was purified from 10-fold serial dilutions of Jurkat cells (5 x 105 to 5 cells) using the RNeasy Plus Micro Kit; at each dilution, RNA was purified from 4 replicates. β-actin transcript was detected in down to 5 cells by real-time RT-PCR, and reproducible CT values were observed at each dilution. In control reactions without reverse transcriptase (-RT), β-actin DNA was not detected after 38 cycles, indicating the absence of genomic DNA contamination.

Specifications

Features	Specifications
Integrated removal of genomic DNA	Yes
Format	Spin column
Technology	Silica technology
Processing	Manual (centrifugation)
Yield	Varies
Elution volume	14 µl
Sample amount	5 x 10e5 cells or 5 mg tissue
Main sample type	Tissue, cells
Applications	Northern, dot, and slot blotting, end-point RT-PCR, quantitative, real-time RT-PCR, array analysis, next-generation sequencing
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein	Total RNA
Time per run or per prep	25 min

Resources

FAQ

I received one of the kits in the list below 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? 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

The MinElute spin columns should be stored at 2-8°C upon arrival. 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

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

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

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

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

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

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.

FAQ ID -1734

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

In addition to all the components included in Buffer RLT, Buffer RLT Plus — a component of RNeasy Plus Kits — also contains a proprietary blend of detergents. These detergents support the efficient binding of DNA molecules to the gDNA Eliminator column or the gDNA Eliminator plate. The exact composition of Buffer RLT Plus is confidential. Buffer RLT Plus can be purchased separately (cat. no. 1053393).

FAQ ID -2794

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

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.

FAQ ID -1750

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

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.

FAQ ID -1749

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

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

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

We do not sell the gDNA eliminator columns separately.

FAQ ID - 3390