miRNeasy Tissue/Cells Advanced Kits

For phenol-free purification of micro RNA and total RNA from tissues and cells, including small samples.

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miRNeasy Tissue/Cells Advanced Micro Kit (50)

Cat. No. / ID: 217684

For 50 preps: RNeasy UCP MinElute spin columns, gDNA Eliminator Spin Columns, Collection Tubes, RNase-Free Water and Buffers

Column typePlate type

Micro

Mini

96 well

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miRNeasy Tissue/Cells Advanced Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

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

Product Details

The miRNeasy Tissue/Cells Advanced Kits are the second generation of the well-established miRNeasy Kits. These improved versions combine a phenol-free advanced chemistry with gDNA Eliminator Columns or gDNA 96 Eliminator Plates for safe and convenient RNA isolation. They enable purification of total RNA, including small RNAs ≥18 nucleotides, from animal tissues and cells, including small samples. The kits use spin columns for low-throughput RNA purification and can be automated on the QIAcube Connect.

Performance

The miRNeasy Tissue/Cells Advanced Kits are phenol-free alternatives to classical miRNeasy Kits without compromising on RNA yields and quality (see figures "High recovery of miRNA without the need for phenol", " High recovery of miR16 from tissues and cells without the need for phenol using miRNeasy Tissue/Cells Advanced Micro", and " High recovery of miRNA without phenol using the miRNeasy 96 Tissue/Cell Advanced Kit"). The miRNeasy Tissue/Cells Advanced Kits enable efficient enrichment of RNA down to approximately 18 nucleotides in size.

The miRNeasy Tissue/Cells Advanced Micro Kit efficiently purifies RNA from up to 5 mg frozen tissue or 1 x 10⁶ cells but also works with significantly lower amounts of starting material. The miRNeasy Tissue/Cells Advanced Mini Kit efficiently purifies RNA from up to 30 mg frozen tissue or 1 x 10⁷ cells. Up to 50 mg tissue and 1 x 10⁷ cells can be processed with the 96-well miRNeasy 96 Tissue/Cell Advanced Kit.

The miRNeasy Tissue/Cells Advanced Kits include the gDNA Eliminator procedure allowing high, reproducible RNA yields and efficient genomic DNA elimination for sensitive applications (see figures " The miRNeasy Tissue/Cells Advanced Mini Kit shows higher performance," " Total RNA extraction: effective genomic DNA removal", and "gDNA 96 Eliminator plates ensure efficient genomic DNA removal"). Total RNA with Agilent RIN or QIAxcel Connect System RIS values close to 10 is routinely obtained from tissues and cultured cells. Unlike kits from other suppliers, the miRNeasy Tissue/Cells Advanced Kits combine ease-of-use and a phenol-free protocol without any compromise on RNA quality or yield (See figures " The miRNeasy Tissue/Cells Advanced Mini Kit shows better performance compared to kits from other supplies," " Competitive performance," " Fast procedure," and The miRNeasy 96 Tissue/Cell Advanced Kit offers excellent performance").

See figures

High recovery of miR16 from tissues and cells without the need for phenol.

High recovery of miRNA without phenol using the miRNeasy 96 Tissue/Cell Advanced Kit

The miRNeasy Tissue/Cells Advanced Mini Kit shows higher performance.

Total RNA extraction: effective genomic DNA removal

The miRNeasy Tissue/Cells Advanced Mini Kit shows better performance compared to kits from other supplies.

Competitive performance

Fast procedure

Principle

The miRNeasy Tissue/Cells Advanced Kits combine 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 Kit procedure"). Next, Buffer AL is added to the lysate to optimize gDNA removal by the gDNA Eliminator Spin Column or gDNA 96 Eliminator Plate. 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. In case of the miRNeasy 96 Tissue/Cell Advanced Kit, a Proteinase K step is included to remove proteins. 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 an RNeasy Mini Spin Column (miRNeasy Tissue/Cells Advanced Mini), an RNeasy UCP MinElute Spin Column (miRNeasy Tissue/Cells Advanced Micro), or to an RNeasy® 96 Plate (miRNeasy 96 Tissue/Cell Advanced), 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

miRNeasy Tissue/Cells Advanced Kit procedure

Applications

The miRNeasy Tissue/Cells Advanced Kits allow purification of miRNA and total RNA for a variety of applications, including:

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

Features	miRNeasy Tissue/Cells Advanced Micro	miRNeasy Tissue/Cells Advanced Mini	miRNeasy 96 Tissue/Cells Advanced Kit
Applications	NGS, real-time RT-PCR, microarray	NGS, real-time RT-PCR, microarray	NGS, real-time RT-PCR, microarray
Elution volume	Default 20 µl, min. 10 µl	30 µl	70-100 µl, min. 45 µl
Format	Spin column	Spin column	RNeasy 96 Plate
Main sample type	Tissue, cells	Tissue, cells	Tissue, cells
Processing	Manual (centrifugation), automated (QIAcube Connect)	Manual (centrifugation), automated (QIAcube Connect)	Manual (centrifugation or centrifugation/vacuum), automated (QIAcube HT)
RNA type	miRNA, total RNA	miRNA, total RNA	miRNA, total RNA
Sample amount	Up to 5 mg tissue, up to 1 x 10⁶ cells	Up to 30 mg frozen tissue (15 mg stabilized) or up to 1 x 10⁷ cells	Up to 30 mg frozen tissue (15 mg stabilized) or up to 1 x 10⁷ cells

Supporting data and figures

miRNeasy Tissue/Cells Advanced Kit procedure

A fast and phenol-free workflow enables microRNA and total RNA isolation including genomic DNA removal.

High recovery of miRNA without the need for phenol

Resources

FAQ

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

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

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

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

Cells do not contain as much proteins as tissue does, therefore a removal of proteins before RNA isolation is not necessary.

FAQ ID-3752

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

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

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

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

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

This is to optimize gDNA removal.

FAQ ID-3751