RNEASY KITS User Manual

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RNeasy Mini Handbook 10/2019

 RNAse/S1 nuclease protection

 Microarrays

The RNeasy Kits allow the parallel processing of multiple samples in less than 30 min.

Time-consuming and tedious methods, such as CsCl step-gradient ultracentrifugation and

alcohol precipitation, or methods involving the use of toxic substances, such as phenol and/or

chloroform, are replaced by the RNeasy procedure.

Principle and procedure

RNA purification using RNeasy technology

The RNeasy procedure represents a well-established technology for RNA purification. This

technology combines the selective binding properties of a silica-based membrane with the

speed of microspin technology. A specialized high-salt buffer system allows up to 100 µg of

RNA longer than 200 bases to bind to the RNeasy silica membrane. Biological samples are

first lysed and homogenized in the presence of a highly denaturing guanidine-thiocyanate–

containing buffer, which immediately inactivates RNAses to ensure purification of intact RNA.

Ethanol is added to provide appropriate binding conditions, and the sample is then applied

to an RNeasy Mini spin column, where the total RNA binds to the membrane and contaminants

are efficiently washed away. High-quality RNA is then eluted in 30–100 µl water.

With the RNeasy procedure, all RNA molecules longer than 200 nucleotides are purified. The

procedure provides an enrichment for mRNA since most RNAs <200 nucleotides (such as 5.8S

rRNA, 5S rRNA, and tRNAs, which together comprise 15–20% of total RNA) are selectively

excluded. The size distribution of the purified RNA is comparable to that obtained by

centrifugation through a CsCl cushion, where small RNAs do not sediment efficiently. Protocols

for purification of small RNA using RNeasy Kits are available at

www.qiagen.com/goto/microRNAprotocols.

In this handbook, different protocols are provided for different starting materials. The protocols

differ primarily in the lysis and homogenization of the sample and in the adjustment of the

conditions for binding RNA to the RNeasy membrane. Once the sample is bound to the

membrane, the protocols are similar (see

Figure 1, page 10).

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User Manual - Page 9