RNeasy Micro Kit

For purification of up to 45 µg total RNA from cell and tissue samples

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 very small amounts of starting material
  • No phenol/chloroform extraction
  • No CsCl gradients, no LiCl or ethanol precipitation
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
The RNeasy 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 Micro Kit is designed for purification of up to 45 μg RNA from small cell and tissue samples. Tissue samples can be conveniently stabilized using RNAprotect Tissue Reagent or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor or TissueLyser system. RNA purification using the RNeasy Micro Kit can be fully automated on the QIAcube Connect. For larger samples, the RNeasy Mini Kit (spin-column binding capacity of 100 µg RNA), RNeasy Midi Kit (spin-column binding capacity of 1 mg RNA), and RNeasy Maxi Kit (spin-column binding capacity of 6 mg RNA) are also available.

Performance

The RNeasy Micro Kit delivers highly reproducible yields of total RNA from small 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"). 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").
See figures

Principle

The RNeasy Micro Kit is designed for isolation of total RNA from small 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 by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification and the unique design of RNeasy MinElute spin columns enables minimal elution volumes, as little as 10 µl, to concentrate the RNA. The small elution volume means that reaction volumes can be kept small in downstream applications, giving increased reaction efficiency. The high purity allows the complete sample to be used in downstream reactions, such as reverse transcription, without enzyme inhibition.

Procedure

The RNeasy Micro Kit is designed for isolation of total RNA from small samples. Samples (<5 mg tissue, or <5 x 105 cells) 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 MinElute spin column and RNA (up to 45 µg) binds to the silica membrane. RNase-free DNase, provided in the RNeasy Micro Kit, enables simple and efficient on-column digestion of genomic DNA for sensitive applications. Then DNase and any contaminants are efficiently washed away, and pure, concentrated RNA is eluted in 10–14 µl water.

Applications

Highly reproducible isolation of RNA in a small elution volume (10 µl) using the RNeasy Micro Kit makes the highly pure RNA well-suited for sensitive quantitative gene expression analyses, including real-time RT-PCR starting with as little as one cell.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsNorthern, dot, and slot blotting, end-point RT-PCR, quantitative, real-time RT-PCR, array analysis, next-generation sequencing
YieldVaries
Elution volume10–14 µl
Purification of total RNA, miRNA, poly A+ mRNA, DNA or proteinRNA
Time per run or per prep30–40 minutes
FormatSpin column
ProcessingManual
TechnologySilica technology
Main sample typeTissue, cells
Sample amount<5 mg

Resources

Kit Handbooks (2)
RNeasy Micro Handbook
PDF (704KB)
For purification of total RNA from small samples, including
  • animal and human cells
  • animal and human tissues
  • fibrous tissues
  • microdissected cryosections
and for RNA cleanup and concentration
RNeasy Micro Handbook
PDF (704KB)
Quick-Start Protocols (1)
RNeasy Micro Kit (EN)
PDF (461KB)

Publications

Stroke induces gene profile changes associated with neurogenesis and angiogenesis in adult subventricular zone progenitor cells.
Liu XS; Zhang ZG; Zhang RL; Gregg S; Morris DC; Wang Y; Chopp M;
J Cereb Blood Flow Metab; 2006; 27 (3):564-74 2006 Jul 12 PMID:16835628
Fibre type distribution and gene expression levels of both succinate dehydrogenase and peroxisome proliferator-activated receptor-gamma coactivator-1alpha of fibres in the soleus muscle of Zucker diabetic fatty rats.
Adachi T; Kikuchi N; Yasuda K; Anahara R; Gu N; Matsunaga T; Yamamura T; Mori C; Tsujimoto G; Tsuda K; Ishihara A;
Exp Physiol; 2006; 92 (2):449-55 2006 Dec 7 PMID:17158179
The phenotype of limbal epithelial stem cells.
Figueira EC; Di Girolamo N; Coroneo MT; Wakefield D;
Invest Ophthalmol Vis Sci; 2007; 48 (1):144-56 2007 Jan PMID:17197527
Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing.
Barbaro V; Di Iorio E; Ferrari S; Bisceglia L; Ruzza A; De Luca M; Pellegrini G;
Invest Ophthalmol Vis Sci; 2006; 47 (12):5243-50 2006 Dec PMID:17122109

FAQ

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
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
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
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
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
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
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 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
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
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
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
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
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
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.
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
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
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 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
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