Principle

Parallel isolation of RNA, DNA, and protein from undivided samples


Simultaneous RNA, DNA, and protein extraction from the same experimental sample is a great and fast growing demand for researchers interested in gene regulation mechanisms, like knock-down of mRNA expression by siRNAs and influences on protein expression. Usually different aliquots of sample material are used to isolate the nucleic acids and protein. This is difficult when working with small, rare, or precious samples. Moreover, the isolation of RNA, DNA, and protein from different sample aliquots usually necessitates proof of sample equality because the analysis might be mistrusted if the experimental sample for nucleic acid and protein purification is not absolutely identical. These issues can be solved by parallel RNA, DNA, and protein isolation from unsplit samples. The NucleoSpin® TriPrep kit allows such a parallel isolation in one working procedure (see procedure). RNA, DNA, and protein are isolated without splitting the sample prior to extraction and are obtained in separate fractions.

Samples are lysed by incubation in a buffer, containing large amounts of chaotropic salts. This will not only destroy the cell structure of the sample but simultaneously inactivate enzymes like, RNases, phosphatases, and proteases and prevent nucleic acids and proteins from degradation. Even hardly soluble proteins are dissolved virtually quantitatively in this lysis buffer. At the same time the buffer creates appropriate binding conditions for RNA and DNA. Thus, nucleic acids and protein from the sample can be separated by simply centrifuging the lysate through the NucleoSpin® TriPrep spin column: RNA and DNA are bound to the silica membrane while the protein is recovered in the column flow-through (see procedure).

The nucleic acids are separated by sequential elution steps. First the DNA is eluted with DNA Elute, a low ionic strength buffer while RNA is still bound to the column. Subsequently RNA is isolated, with wash steps, on-column DNA digestion (to remove residual DNA contaminations), and elution of high quality RNA. Proteins in the flow-through are precipitated by a special buffer (Protein Precipitator), pelleted by centrifugation, washed, and finally dissolved in Protein Solving Buffer.
Isolated RNA and DNA are of high quality and directly suitable for all common downstream applications. Isolated protein is immediately suitable for quantification, SDS-PAGE, and Western blot analysis. 

Features

*Binding capacity of DNA ≤ 10 μg, strongly depending on RNA amount bound to the membrane.