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RNeasy Lipid Tissue Midi Kit

For purification of up to 1 mg total RNA from fatty tissues and other types of tissue
  • Optimized lysis conditions for fatty tissues and other tissues
  • High yields of total RNA without phenol contamination
  • High-quality RNA for all downstream applications
The RNeasy Lipid Tissue Midi Kit includes QIAzol Lysis Reagent for lysing fatty tissues and other types of tissue, and RNeasy spin columns for purifying up to 1 mg of high-quality RNA. Tissue samples can be conveniently stabilized using RNAlater RNA Stabilization Reagent (nonfatty tissues only) or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor or TissueLyser system. For smaller samples, the RNeasy Lipid Tissue Mini Kit (spin-column binding capacity of 100 µg RNA) is also available.
The RNeasy Lipid Tissue Midi Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
1
High yields of RNA without phenol carryover.
RNA was isolated from 10 mg rat brain tissue using the RNeasy Lipid Tissue Mini Kit (RNeasy Lipid), a standard silica-gel-membrane procedure (Std. silica), or a phenol/guanidine-based reagent (PhOH/Gdn), following supplier's instruction. [A] Formaldehyde agarose gel analysis shows high yields of RNA using the RNeasy Lipid Tissue Mini Kit. Using the RNeasy and other silica-based methods, small RNAs (such as 5.8S rRNA, 5S rRNA, and tRNAs) are selectively excluded. [B] Absorbance spectrum shows contaminants and phenol when using the phenol/guanidine-based reagent.
2
Real-time analysis.
Total RNA was isolated from 100 mg rat brain or adipose tissues using the RNeasy Lipid Tissue Mini Kit. Real-time, quantitative RT-PCR was carried out using QuantiTect Probe RT-PCR Kit with the indicated amounts of total RNA (in duplicate) and primers and probe specific for the c-jun or alpha-actin genes.
3
RNeasy Lipid Tissue Midi procedure.

The RNeasy Lipid Tissue Midi kit integrates phenol/guanidine-based lysis with RNeasy silica-membrane purification for isolation of high-quality total RNA. After centrifugation, RNA partitions to the upper, aqueous phase, while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase. Ethanol is added to the aqueous phase, and the sample is applied to the RNeasy spin column.

Performance
RNeasy Lipid Tissue Kits are optimized for use with fatty tissues with RNA yields from 100 mg of representative fatty tissues of 110 µg from brain, and 21 µg from adipose tissue. The RNeasy Lipid Tissue procedure ensures easy and efficient isolation of high-quality total RNA without phenol carryover (see figures "High-quality RNA" and "High yields of RNA without phenol carryover"). RNA purified with the kits from lipid tissue is suitable for real-time RT-PCR (see figure "Real-time analysis").
Principle

RNeasy Lipid Tissue Kits are optimized for use with fatty tissues, such as brain and adipose tissue. These kits integrate optimized phenol/guanidine-based lysis with proven RNeasy silica-membrane purification for isolation of high-quality total RNA. The combination of organic extraction and chaotropic disruption contributes to the high lysis efficiency of QIAzol Lysis Reagent, enabling use of larger amounts of fatty tissues in the RNeasy procedure for high yields of total RNA. Since the RNeasy procedures enrich for mRNA and other RNA species >200 nucleotides, the total RNA yield does not include 5S rRNA, tRNA, and other low-molecular-weight RNAs, which make up 15-20% of total cellular RNA.

Procedure

Tissue samples 75–500 mg are homogenized in QIAzol Lysis Reagent. After addition of chloroform, the homogenate is separated into aqueous and organic phases by centrifugation. The upper, aqueous phase is extracted, and ethanol is added to provide appropriate binding conditions. The sample is then applied to the RNeasy spin column, where the total RNA up to 1 mg binds to the membrane and phenol and other contaminants are efficiently washed away. High-quality RNA is then eluted in 150–500 µl RNase-free water water (see figure "RNeasy Lipid Tissue Midi procedure").

Applications

RNeasy Lipid Tissue Kits provide easy and efficient isolation of high-quality RNA for all downstream applications, including array analysis and real-time RT-PCR.

Features
Specifications
Applications PCR
Elution volume 150–500 µl
Format Spin column
Main sample type Fatty tissue samples
Processing Manual
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein RNA
Sample amount 75–500 mg
Technology Silica technology
Time per run or per prep 60 minutes
Yield 21–110 µg
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