Welcome to the newest version of the Biospecimen Research Database (BRD), which accommodates Standard Operating Procedures (SOP). We encourage your contributions to our new SOP library!
The BRD is a free and publicly accessible database that contains peer-reviewed primary and review articles as well as SOPs in the field of human Biospecimen Science. Each literature curation has been created by a Ph.D.-level scientist to capture the following: (1) relevant parameters that include the biospecimen investigated (type and location, patient diagnosis), preservation method, analyte(s) of interest and technology platform(s) used for analysis; (2) the pre-analytical factors investigated, including those relating to pre-acquisition, acquisition, preservation, processing, storage, and analysis; and (3) an original summary of relevant results. Browse literature curations or submit specific queries using the Advanced Search page with keyword search for specific biomakers or genes, PubMed ID, or pre-analytical factor values (anticoagulant, fixative, reagent, etc).
SOPs are organized in a hierarchy system consisting of two tiers: (1) SOPs, established protocols; and (2) Biospecimen Evidence-based Practices (BEBP), procedural guidelines developed using literature evidence. SOP-tiered documents are a product of the Source organization specified. SOPs shared by external organizations are done so only with their consent, and have not been vetted by BBRB. SOP documents are searchable by keyword, or by curated fields (source organization, tier, applicable biospecimens, and topic) on the Search SOPs page. Related SOP documents are assembled in Compendiums, which are viewable on the SOP Compendiums page. You can also create your own Compendium and download SOPs together rather than individually.
We encourage you to submit SOPs from your lab or institution for inclusion in the BRD by clicking on the Submit an SOP tab or at email@example.com. Individuals and organizations that suggest articles for inclusion in the BRD will receive acknowledgement on the paper's curation page. Articles may be submitted by clicking on the Suggest a New Paper tab or via the email above. Feedback is also welcome.
The BRD is an initiative of the NCI Biorepositories and Biospecimen Research Branch (BBRB).
The purpose of this study was to compare DNA yield, purity, and integrity and NGS data from FFPE, PFPE, and RNAlater preserved specimens and to compare results using different DNA extraction methods. The DNA yield and integrity assays were used to develop a DNA quality score that predicted NGS success. Matched FFPE, PFPE, and RNAlater-preserved specimens from three breast tumors, three normal colons, three normal skin specimens, two kidney tumors, one normal kidney, one uterine tumor, and two normal uterus specimens (15 specimens total). All tumor specimens contained 40-90% tumor, 5-10% normal tissue, and <20% necrosis. The authors report specimen preservation was according to manufacturers’ protocols. DNA was extracted from RNAlater preserved specimens using the DNeasy Blood &Tissue kit. DNA was extracted from two to three 20µm scrolls of FFPE and PFPE specimens by: (1) deparaffinization in xylene followed by two absolute ethanol washes and extraction using the DNeasy Blood & Tissue Kit or (2) deparaffinization in Chemagic lysis buffer at 95°C followed by extraction using the Chemagic DNA Tissue Kit special: from FFPE specimens by (3) deparaffinization using three xylene washes, two methanol washes and a graded ethanol series followed by the QIAamp DNA FFPE Tissue Kit, and from PFPE specimens using (4) a single xylene wash followed by a single absolute ethanol wash and extraction with PAXgene Tissue DNA kit. DNA concentration was determined spectrophotometrically and using Quant-IT PicoGreen dsDNA Assay Kit, purity was evaluated spectrophotometrically, and integrity was assessed by GAPDH multiplex PCR (100, 200, 300, and 400 bp). DNA integrity was also assessed based on the yield using the BIOSCORE Screening and Amplification Kit with DNA scored as poor (<1 µg generated) intermediate (1-3 µg generated), good (3-10 µg generated), or excellent (>10 µg generated). Targeted TruSeq Amplicon Cancer Panel libraries were constructed following the Illumina protocol using 150 ng DNA (FFPE specimens) or 250 ng DNA (RNAlater and PFPE specimens) from eight patients and sequenced using a MiSEq instrument. Analysis was conducted using the Illumina Variant Studio software. Acceptance for the run was defined as having a global Q score of 30-80%, a raw cluster density between 500-1000 k/mm2, and >85% of clusters passing filters. Samples were considered acceptable if Phred score was >49, read depth was >999, and alternate variant frequency was >5%.
|Technology Platform||Analyte||Spectrophotometry||DNA||Fluorometry||DNA||Next generation sequencing||DNA||Whole genome amplification||DNA||PCR||DNA|
The top 5 most downloaded SOPs in the BRD in 2018 were supplied by:
GAPPS Repository: https://brd.nci.nih.gov/brd/sop/show/890
A new article from NCI's BPV Program is now available that reports deleterious effects of formalin-fixation and delays to fixation on RNA and miRNA-Seq profiles.
View the PubMed Abstract at https://www.ncbi.nlm.nih.gov/pubmed/31061401More...