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Hospital Universitario de Jaen

A sustainable alternative for DNA preservation.

DNA is a naturally stable molecule that is well suited to long-term storage. However, inadequate storage makes it susceptible to degradation processes that can compromise its quality—especially when it comes to meeting the demands of new and more sensitive molecular biology techniques.

As a result, despite its supposed stability, DNA is still routinely stored in ultra-low temperature freezers, occupying between 10% and 20% of available storage capacity in biobanks and laboratories.

Objective

Compare the integrity, purity, and functionality of DNA extracted from paraffin and subsequently lyophilised, comparing it with the same DNA frozen at -80°C.

Materials and Methods

Samples used for the study

DNA samples

15 samples

Frozen

DNA samples

15 samples

FFPE

Procedure

Thawed DNA will be selected from samples with an initial DIN >3 and sufficient quantity to proceed with the study. The DNA obtained from paraffin blocks will be measured using a Qubit to calculate the quantity and integrity. If the DIN is greater than 3, the sample will be included in the study. These samples will undergo NGS to detect mutations.

Each sample is then divided into two aliquots: one aliquot is labeled and stored using the conventional method at −80 °C, while the other is labeled and freeze-dried using the S3 device. Frozen samples included in the study are first thawed prior to freeze-drying.

After completion of the lyophilization cycle and verification that a proper pellet has formed, all freeze-dried samples are stored at room temperature in the appropriate storage boxes. Following storage periods of 1, 6, and 12 months under desiccated conditions, freeze-dried samples are re-evaluated and directly compared with their corresponding frozen counterparts.

Quality Assurance

DNA Concentration

DNA integrity number

EGFR, RAS and B-RAF gene mutations

Sequencing

All samples processed and stored under the different conditions (freeze-dried or frozen) will be analyzed simultaneously to minimize inter-assay variability. The following techniques will be used for quality assessment:
  • Quantification of double-stranded DNA using the Qubit® fluorometric quantification system.
  • Assessment of DNA integrity using the Genomic DNA (gDNA) ScreenTape assay on the TapeStation system.
  • EGFR, RAS and B-RAF gene mutations by PCR (Frozen samples underwent PCR testing at diagnosis to detect mutations in genes such as EGFR, RAS and BRAF, depending on whether the cancer was in the lungs or colon. The same PCR test will be repeated after lyophilisation to check for any changes.)
  • Additionally, the Hospital’s Anatomy Service may perform sequencing studies on some of the samples. (The freshly extracted samples will undergo NGS to detect mutations. This process will be repeated after lyophilisation.)

Results

Coming soon… 

Conclusion

Coming soon… 

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