EZ2 Connect Kit for Automated Total RNA Extraction

For automated purification of total RNA, including small RNAs, using EZ2 Connect instruments

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EZ2 RNA/miRNA Tissue/Cells Kit (48)

Cat. No. / ID: 959035

For 48 preps: EZ2 RNA/miRNA Tissue/Cells cartridge, filter tips and holders, tubes, RNase-free DNase, Buffer RLT, Proteinase K

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The EZ2 RNA/miRNA Tissue/Cells Kit is intended for molecular biology applications. This product is 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

Purifies RNA and miRNA from human and animal cells and tissues
Does not use toxic phenol
Includes highly efficient enzymatic removal of protein and gDNA
Lets you process up to 24 samples at the same time, for easy intra-run comparability
Prevents RNase contamination with prefilled cartridges and closed workdeck

Product Details

The EZ2 RNA/miRNA Tissue/Cells Kit makes it easy and safe to purify total RNA – including miRNA and other small RNA – from cultured cells and various human or animal tissues. The process requires no use of phenols, and once the lysis step is completed, all remaining steps are automated with any EZ2 Connect instrument.

Performance

The EZ2 RNA/miRNA Tissue/Cells protocol isolates a similar amount of total RNA, including small RNA, to manual column-based protocols. This is true even for difficult tissue types such as fibrous tissue (e.g., muscle) or fatty tissue (e.g., brain) (see Comparable miRNA recovery). Genomic DNA removal is likewise comparable to manual column-based purification (see Efficient mRNA recovery and gDNA removal). Digital PCR analysis shows higher RNA concentration in samples processed with the EZ2 RNA/miRNA Tissue/Cells Kit (see Higher RNA concentration).

See figures

Comparable miRNA recovery.

Efficient mRNA recovery and gDNA removal.

Higher RNA concentration.

Principle

The EZ2 RNA/miRNA Tissue/Cells protocol uses manual lysis in Buffer RLT protect the RNA molecules. Proteinase K digestion ensures complete lysis of even hard-to-lyse tissues. Bind, wash, and elute steps are automated on an EZ2 Connect instrument, which uses magnetic bead technology. After first binding, RNA is treated with DNase to digest contaminating genomic DNA, and then rebound to the magnetic beads. Wash steps then remove contaminants that could interfere with enzymatic reactions that follow.

Procedure

The cell pellet or cell culture is first homogenized, or the tissue sample is disrupted and then homogenized. RNase-free water and Proteinase K are then added, and the mixture is incubated for 10 minutes at room temperature (see EZ2 RNA/miRNA Tissue/Cells workflow).

After incubation, samples and prefilled reagent cartridges are loaded into the EZ2 Connect, and application run is started. Once the run is finished, purified RNA or miRNA will be ready for downstream applications (see EZ2 RNA/miRNA Tissue/Cells workflow).

See figures

EZ2 RNA/miRNA Tissue/Cells workflow.

Applications

RNA and miRNA purified with the EZ2 RNA/miRNA Tissue/Cells Kit is highly suitable for RT-PCR, digital PCR (dPCR) or next-generation sequencing (NGS).

Supporting data and figures

EZ2 RNA/miRNA Tissue/Cells workflow.

Disrupt and homogenize the sample. Lyse with Proteinase K. Transfer sample to the EZ2 Connect instrument. Start the run. When the run is finished, retrieve purified RNA or miRNA elute.

Specifications

Features	Specifications
Applications	RT-PCR, dPCR, NGS
Elution volume	50 or 100 µl
Main sample type	Cells, tissue
Processing	Automated with an EZ2 Connect instrument
Analyte	Total RNA, including miRNA and mRNA
Sample amount	Up to 30 mg (frozen) or 15 mg (stabilized) tissue or 10 mg frozen spleen or lung tissue, or up to 5 x 10^6 cells
Technology	Magnetic-particle technology

Resources

For automated purification of total RNA including small RNAs using EZ2 Connect instruments

For use with EZ2 Connect instruments

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

All versions of the EZ2 Connect (EZ2 Connect, EZ2 Connect Fx, and EZ2 Connect MDx) can run the kit.

FAQ ID - 3851

No, everything is included in the kit box.

FAQ ID - 3849

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

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

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

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

No, it is only compatible with EZ2 Connect.

FAQ ID - 3852

Yes, this is possible to some extent. The SAP number for "customized protocols" will be implemented.

FAQ ID - 3850