Bio-Rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit - Manual (Page 5)

Section 1
Introduction
The Aurum™ total RNA fatty and fibrous tissue kit produces high yields of
pure total RNA from samples that are difficult to disrupt. The kit is ideal for
fatty or fibrous tissues, or samples that are rich in RNases. It also works well
with most animal and plant tissues, cultured cells, yeast, and gram-negative
and gram-positive bacteria. With this kit, greater than 100 µg of total RNA can
be isolated from many sample types. Total RNA samples isolated using the
Aurum total RNA fatty and fibrous tissue kit are suitable for use in a variety of
downstream applications, including reverse transcription-PCR (RT-PCR), real-
time PCR, in vitro translation, northern blots, and microarray analysis.
The Aurum total RNA fatty and fibrous kit uses a quick, easy-to-follow
procedure for the purification of total RNA from difficult-to-disrupt samples.
First, samples are disrupted and lysed using the PureZOL™ RNA isolation
reagent. Subsequently, chloroform is added to the lysate, mixed, and
centrifuged to achieve separation of the organic and aqueous phases. The
aqueous phase, which contains the RNA, is carefully recovered and mixed
with ethanol. The sample is then passed through a silica membrane packed in
the Aurum RNA binding mini column, where nucleic acids get bound. Wash
steps are performed to remove proteins and other cellular debris. An optional
on-column DNase I digest is performed to remove any remaining genomic
DNA. The RNA, which is eluted with the elution solution supplied in the kit, is
now ready for downstream applications without further manipulation.
The membrane in the Aurum RNA binding mini column selectively binds to
mRNA and larger rRNAs, while small RNA molecules less than
200 nucleotides, such as 5.8S rRNA, 5S rRNA, and tRNA (which together
comprise 15–20% of total RNA), are removed. The on-column DNase l digest
and subsequent wash steps that are performed during the purification
effectively remove genomic DNA contamination as well as the residual DNase
enzyme, eliminating the need for separate and lengthy DNase treatments and
DNase removal protocols on the eluted RNA.
The Aurum total RNA fatty and fibrous tissue kit is designed to isolate total
RNA from various amounts of tissue (5–100 mg) and cells (50–2.4 x 10
9
cells)*. The kit includes sufficient reagents and columns for 50 purifications. All
solutions and RNA binding mini columns in the kit are RNase-free, ensuring
the integrity of the isolated RNA. The Aurum total RNA fatty and fibrous tissue
kit may be used in a spin format, or in a vacuum format using the Aurum
vacuum manifold (catalog # 732-6470).
* The amount of starting material may be less for certain types of samples. For more detail, refer to
Section 5.
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