eBook: Optimizing Results from Nucleic Acid Isolation - 21

For RNA isolation, guanidium thiocyanate or a similar chaotropic agent is used to lyse the cells and
denature RNase and DNase enzymes, protecting
the sample(s). Once added, the samples require robust shaking, usually vortexing, in the presence of
a reducing agent, such as β-mercaptoethanol. This
process breaks any disulphide bonds and inactivates
any contaminant proteins present in the sample(s).

NGS, and quantitative real-time PCR (qRTPCR), making them an invaluable resource for molecular biology and genetic studies.
There are many protocols available to process fresh
frozen tissue samples, including manual methods
developed in-house and a variety of commercial
kits. These methodologies are rapid and straightforward, requiring thawing and mechanical disruption of the tissue samples in the presence of a chaotropic lysis buffer.

Importantly, and usefully for researchers, DNA and
RNA can both be isolated from the same biological
sample. This process involves extracting a total nucleic acid fraction and dividing it into two. One half is
treated with DNase 1, and the other RNase A.

Another invaluable source of material, FFPE tissues,
are held as large archives in pathology departments
and clinical laboratories around the world.

Fresh frozen and formalin-fixed
paraffin embedded samples in
nucleic acid isolation

FFPE blocks enable the prolonged storage of clinical samples for investigation, preserving the tissue
morphology for pathological examination, cancer diagnosis, and nucleic acids for isolation and
downstream molecular analysis. These samples are
easy to collect and of great interest to laboratories
conducting clinical genomic studies, transcriptome
analysis, biomarker identification, and chemo-resistance research.

Fresh frozen tissue samples are widely considered
the easiest type of tissue sample to process for nucleic acid isolation. The absence of chemicals and
rapid freezing means that the nucleic acids, as well
as the proteins within them, remain unmodified and
in their native state(s).

The quality and integrity of the DNA and RNA from
FFPE samples are affected by a number of factors1, 2:

This approach means fresh frozen tissue samples are
well suited for mass spectrometry, western blotting,

* pH of the fixative.
* Length of tissue fixation.
* Age and storage condition of tissue blocks.
* Extraction method used.

Addressing the challenges with fresh
frozen and FFPE samples
Despite the ease of using fresh frozen and FFPE samples, there are challenges. For fresh frozen, the most
substantial challenge is temperature. The samples
rapidly deteriorate at room temperature. At the point

eBook: Optimizing Results from Nucleic Acid Isolation

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