The RNeasy Midi Kit allows efficient purification of total RNA from medium amounts of starting material. Total RNA is easily purified from animal or human cells, animal or human tissues, and yeast* (see table "Total RNA yields obtained with the RNeasy Midi Kit" and figure "High-quality RNA from a variety of samples"). Protocols are also included for isolation of cytoplasmic RNA^† from eukaryotic cells as well as for cleanup of partially purified RNA, in vitro transcripts, and RNA from enzymatic reactions. A variety of special application protocols are also available.

Specifications of the RNeasy Midi Kit

Specification	Details
Format	Midi spin columns with 15 ml collection tubes
Sample size	5 x 106 – 1 x 108 cells 20–250 mg tissue 2 x 107 – 5 x 108 yeast cells
Preparation time	<1 hour
Binding capacity	Up to 1 mg RNA
Yield	Varies depending on sample source. See table.
Elution volume	300–500 µl

RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification. There is no need for centrifugation through CsCl cushions or precipitation with LiCl or alcohol. RNeasy sample preparation technology is fully licensed, allowing RNeasy purified nucleic acids to be used in any molecular assay or other downstream application without risk of patent infringement.

RNeasy Kits provide the highest-quality RNA with minimum copurification of DNA. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using the RNase-Free DNase Set for convenient on-column DNase treatment during the RNeasy procedure.

Total RNA yields obtained with the RNeasy Midi Kit^‡

Source	Average yield (µg)
Animal cells LMH (7 x 107) HeLa (7 x 107) COS-7 (3 x 107) Lymphocytes (unstimulated) (1 x 108)	850 1000 950 50
Mouse tissue Liver (200 mg) Lung (100 mg) Spleen (200 mg)	700 130 600
Yeast cells S. cerevisiae (2 x 108)	450

* Lyticase, zymolase, or glass beads (required for yeast samples) are not provided.
^† Additional buffer required. The buffer composition is provided in the protocols.
^‡ Amounts can vary due to developmental stage, species, growth conditions used, etc. Since the RNeasy procedure enriches for RNA species >200 nt, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs.

Procedure

Samples 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 silica membrane (see figure "RNeasy Midi spin column"). RNA binds, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in water.

Applications

RNA purified with RNeasy technology has A260/A280 ratios of 1.9–2.1 (measured in 10 mM Tris·Cl, pH 7.5) and is ideal for use in all applications. Downstream applications include:

• Northern, dot, and slot blotting
• End-point RT-PCR
• Quantitative, real-time RT-PCR
• Array analysis
• Poly A+ RNA selection