Table 1.
Relevant biospecimen information recorded on the sample tracking sheet.
Table 2.
Critical parameters and acceptance criteria for sample management process validation and verification.
Table 3.
Initial and optimized conditions for plasma and serum biospecimen preparation and the corresponding findings.
Fig 1.
DNA integrity according time of storage in the biobank.
After extraction, DNA was separated on 0.8% agarose gel and visualized by Gel Red staining on an imager. (A) Electrophoresis of increasing amounts of DNA after extraction from PAXgene tube. (B) Electrophoresis of DNA after extraction and storage at –80°C. The year in which the extraction is carried out is indicated. MM: molecular markers.
Table 4.
Quality metrics of DNA isolated from blood.
The concentration and purity of the DNA samples isolated from the patients’ blood were evaluated based on spectrophotometric measurements at 260 nm and 280 nm.
Table 5.
Quality metrics of DNA isolated from blood after destocking from the biobank.
The concentration and purity of the DNA samples isolated from the patients’ blood were evaluated based on spectrophotometric measurements at 260 nm and 280 nm.
Fig 2.
Electropherograms of RNA samples obtained for the four tissue types.
Electropherograms were obtained on RNA samples using 2100 Bioanalyser and RNA 6000 Nano chips. Typical electrophoretic traces and RIN were shown for VAT, SCAT, liver, and muscle extracted in 2016. SCAT, sub–cutaneous adipose tissue; VAT, visceral adipose tissue.
Table 6.
Quality metrics of RNA isolated from frozen tissues obtained from the first five patients included.
Absorbance at 260 and 280 nm, RIN and 28S/18S ratio were assessed for each RNA sample isolated from frozen SCAT, VAT, muscle, and liver. Mean ± SD, median, and ranges for each type of RNA are shown. The number of samples (and percentage) in the optimal value of purity (A260/280 ≥ 2) and integrity (RIN ≥ 7, 28S/18S ratio: 1.9–2.1) is also presented.
Fig 3.
RIN according to handling time for tissue preparation for the first five patients.
RNA was extracted from frozen tissues. RIN was shown according to handling times of liver, muscle, SCAT and VAT samples resected from the first five patients, before snap–freezing. The handling times between tissue resection and snap–freezing were: ≤ 10, ≤ 15 and ≤ 20 minutes (all tissues) and ≤ 25 minutes (adipose tissues only). SCAT, sub–cutaneous adipose tissue; VAT, visceral adipose tissue.
Fig 4.
Cycle threshold (Ct) values of six housekeeping genes according to handling time for tissue preparation for the first five patients.
RNA was used to perform a RT–qPCR in order to amplify six housekeeping genes (ACTB, B2M, GAPDH, HPRT, TBP, 18S). Ct values were shown for the genes according the type of tissue samples resected from the first five patients and the handling time between tissue resection and snap–freezing. VAT, visceral adipose tissue.
Fig 5.
RIN according to the time of sample storage in the biobank.
For each patient, RIN was shown for RNA samples 1) obtained after extraction from the four tissue types carried out in 2016 or 2017, 2) after their storage at –80°C during 3 to 4 years and re–evaluation in 2020, 3) obtained after destocking of frozen tissues from the biobank and extraction in 2019 and 2020. Results are shown for samples obtained from the first five patients included in 2016 and other patients included in 2017. SCAT, sub–cutaneous adipose tissue; VAT, visceral adipose tissue.
Table 7.
Quality metrics of RNA isolated from frozen tissues after different time of storage in the biobank.
Mean RIN ± SD were shown for RNA samples extracted from frozen tissues (SCAT, VAT, liver, and muscle) in 2016, 2017 and for RNA samples obtained from the same tissues and patient in 2019 and 2020. RNA samples extracted in 2016 and 2017 were also re–evaluated in 2020 (“redosing”). Mean DV200 ± SD were shown for RNA samples extracted in 2019 and 2020.
Fig 6.
Histological integrity according to the time of sample storage in the biobank.
Sections of SCAT, liver and muscle stored since 2016 were performed and stained with hematoxylin–eosine in 2020. Images were shown according to the material preparation protocol: (A) direct evaluation on frozen tissue sections (B) evaluation after the tissue was thawed, fixed and embedded in paraffin. Magnification is x200. SCAT, sub–cutaneous adipose tissue.
Table 8.
Histological integrity according to the time of sample storage in the biobank.
Tissue samples from 3 patients were analyzed for their histological integrity after several months of storage in the biobank. Sections of SCAT, liver and muscle were performed directly on frozen tissues or after thawing, fixation and embedding in paraffin, and stained with hematoxylin–eosine. Histological integrity was analyzed by the pathologist under an optical microscope. S: satisfactory quality; NS: not satisfactory; ND: not determined. SCAT, sub–cutaneous adipose tissue.