miRNeasy Tissue/Cells Advanced Mini Kit

For phenol-free purification of microRNA and total RNA from tissues and cells

Features

  • Phenol-free protocol – no need for phase separation or working under a hood
  • Fast and convenient procedure due to the advanced chemistry and gDNA Eliminator Columns
  • Efficient enrichment of miRNA and RNAs <200 nucleotides
  • Consistent, pure RNA for all downstream applications
miRNeasy Tissue/Cells Advanced Mini Kit (50)

Cat. No. / ID: 217604

For 50 preps: RNeasy Mini Spin Columns, gDNA Eliminator Spin Columns, Collection Tubes, RNase-Free Water and Buffers

Product Details

The miRNeasy Tissue/Cells Advanced Mini Kit is the second generation of the well-established miRNeasy Mini Kit. This improved version combines a phenol-free advanced chemistry with gDNA Eliminator Columns for safe and convenient RNA isolation. miRNeasy Tissue/Cells Advanced Mini Kit enables purification of total RNA, including small RNAs ≥18 nucleotides, from animal tissues and cells. The kit uses spin columns for low-throughput RNA purification. Furthermore, the entire procedure can be automated on QIAcube Connect.

Performance

The miRNeasy Tissue/Cells Advanced Mini Kit is a phenol-free alternative to the miRNeasy Mini Kit with similar RNA yields and quality (see figure " High recovery of miRNA without the need for phenol"), and enables efficient enrichment of RNA down to approximately 18 nucleotides in size. The miRNeasy kits efficiently purify RNA from up to 30 mg frozen tissue or 1 x 107 cells, but also works with lower amounts of starting material. The miRNeasy Tissue/Cells Advanced Mini Kit includes the gDNA Eliminator procedure allowing high, reproducible RNA yields and efficient genomic DNA elimination for sensitive applications (see figure " Total RNA extraction: effective genomic DNA removal"). Total RNA with Agilent RIN values close to 10 is routinely obtained from tissues and cultured cells. Unlike kits from other suppliers, the miRNeasy Tissue/Cells Advanced Mini Kit combines ease-of-use and a phenol-free protocol without any compromise on RNA quality or yield (see figures " Competitive performance" and " Fast procedure").

 

See figures

Principle

The miRNeasy Tissue/Cells Advanced Mini Kit combines a guanidine-based sample lysis with an inhibitor removal step and a silica-membrane-based purification of total RNA, which includes miRNA and other small RNA. Buffer RLT, included in the kit, contains guanidine thiocyanate which facilitates lysis and denaturation of RNases and other proteins. Therefore, RNA in samples lysed in buffer RLT are stable and protected from degradation.

Procedure

Sample lysis is achieved by adding Buffer RLT to tissue or cell samples followed by a thorough disruption and homogenization step to ensure complete lysis and inactivation of RNases (see figure " miRNeasy Tissue/Cells Advanced Mini procedure"). Next, Buffer AL is added to the lysate to optimize gDNA removal by the gDNA Eliminator Spin Column. Depending on the type and amount of starting material, an optional on column DNase digestion can be carried out (not included in the kit). Buffer RPP is added to the flow-through to precipitate inhibitors (mostly proteins that are highly concentrated in tissue and cell samples and might interfere with the RNA isolation or the downstream analysis) by centrifugation. The supernatant containing the total RNA is transferred into a new reaction tube, and isopropanol is added to provide appropriate binding conditions for all RNA molecules ≥18 nucleotides. The sample is then applied to a RNeasy Mini Spin Column, where the total RNA binds to the membrane and all contaminants are efficiently washed away. High-quality RNA, including miRNA and other small RNA, are eluted in a small volume of RNase-Free Water.

See figures

Applications

The miRNeasy Tissue/Cells Advanced Mini Kit allows purification of miRNA and total RNA for a variety of applications, including:

  • Quantitative, real-time RT-PCR
  • RNA-seq
  • Microarray analysis
  • Northern blot analysis

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsNext-generation sequencing, real-time RT-PCR, microarray
Elution volume30 µl
FormatSpin column
Main sample typeTissue, cells
ProcessingManual (centrifugation)
Purification of total RNA, miRNA, poly A+ mRNA, DNA or proteinmiRNA, total RNA
Sample amountup to 30 mg frozen tissue (15 mg stabilized), up to 1 x 10e7 cells
TechnologySilica technology
YieldVaries

Resources

Brochures & Guides (1)

FAQ

How much RNA does a typical mammalian cell contain?

The RNA content and RNA make up of a cell depend very much on its developmental stage and the type of cell. To estimate the approximate yield of RNA that can be expected from your starting material, we usually calculate that a typical mammalian cell contains 10–30 pg total RNA.

 

The majority of RNA molecules are tRNAs and rRNAs. mRNA accounts for only 1–5% of the total cellular RNA although the actual amount depends on the cell type and physiological state. Approximately 360,000 mRNA molecules are present in a single mammalian cell, made up of approximately 12,000 different transcripts with a typical length of around 2 kb. Some mRNAs comprise 3% of the mRNA pool whereas others account for less than 0.1%. These rare or low-abundance mRNAs may have a copy number of only 5–15 molecules per cell.

FAQ ID -2946
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
What can I use to isolate RNA smaller than 200 nucleotides?

For the isolation of microRNA (miRNA) specifically, we have developed the miRNeasy Mini Kit and the miRNeasy 96 Kit for isolation from cells and tissues.   We also have the PAXgene Blood miRNA and Tissue miRNA kits for isolation from blood stored in PAXgene Blood RNA tubes and PAXgene Tissue Containers, respectively.  Other miRNA isolation supplementary protocols can also be found by searching our comprehensive protocols at http://www.qiagen.com/literature/default.aspx?WT.svl=m.

FAQ ID -115
What is the composition of Buffer RWT?
The exact composition of Buffer RWT is confidential. Buffer RWT is a proprietary component of, for example, the miRNeasy Mini Kit and the RNeasy Plus Universal Kit. Guanidine salt and ethanol are important ingredients in Buffer RWT. Ethanol is added by the user prior to the first use of the kit. Buffer RWT is a stringent washing buffer used after preclearing the sample with QIAzol Lysis Reagent, especially if isolation of small RNAs, for example, microRNAs or RNAs from formalin-fixed tissue, is desired
FAQ ID -2798
Why is the protein precipitation step optional when working with cells?
Cells do not contain as much proteins as tissue does, therefore a removal of proteins before RNA isolation is not necessary.
FAQ ID-3752
How do I clean up RNA preparations containing miRNA?

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.

FAQ ID -3002
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
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 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
Can I use total RNA for the miRNA PCR Arrays or Assays?
Yes, you can. In fact, total RNA is the recommended starting material for the miScript PCR System. We recommend using the miRNeasy Mini Kit (217004) to isolate total RNA for use with the miScript PCR System.
FAQ ID -2726
Why do I have to add Buffer AL before using the gDNA Eliminator?
This is to optimize gDNA removal. 
 
FAQ ID-3751