Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples.
Author(s): Domínguez-Vigil IG, Barajas-Olmos VH, Gallardo-Alvarado L, Pérez-Maya AA, Garza-Rodríguez ML, Magallanes-Garza GI, Cardona-Huerta S, Méndez-Lozano DH, Vidal-Gutiérrez O, Cantú De León DF, Barrera-Saldaña HA
Publication: Biopreserv Biobank, 2019, Vol. 17, Page 591-597
PubMed ID: 31556699 PubMed Review Paper? No
Purpose of Paper
This paper compared the effects of type of cytological brush used for collection, storage duration, and extraction method used (manual versus automated) on yield, concentration, integrity, and PCR success of DNA from cervical specimens.
Conclusion of Paper
DNA yields were higher when specimens were extracted using the manual method compared to the automated method, regardless of cytological brush type used but yields were not affected by storage duration. DNA concentration, integrity, and amplification success of the 268 bp fragment of the β-globin gene were comparable among collection brush types, extraction methods, and storage durations.
Studies
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Study Purpose
This study compared the effects of type of cytological brush used for collection, storage duration, and extraction method used (manual versus automated) on yield, concentration, integrity, and PCR success of DNA from cervical specimens. Specimens were collected from 296 women (age range: 19–85 y, mean age: 49) between July 2015 and September 2017 using either the Colpotre cytological brush with cells collected by five clockwise rotations or using the Rovers Cervex-Brush-Combi with cells collected by two clockwise rotations. Brushes were placed in a 50 mL conical tube and stored at 4°C until processing (within 1–52 d). Specimens were vortexed for 30 seconds, centrifuged at 300 x g for 10 minutes at 4°C, and pelleted cells were homogenized in the presence of lysis buffer. DNA was extracted with the Qiagen AllPrep DNA/RNA/microRNA Universal Kit using either a manual (n=233) or automated (n=63) method. A sample set of specimens (n=54) were analyzed for DNA integrity by agarose gel electrophoresis. DNA yield was measured by fluorometry, purity determined by A260/280 and A260/230 ratios using a NanoDrop spectrophotometer, and quality was determined by PCR amplification of a 268 bp fragment of the β-globin gene.
Summary of Findings:
DNA yields were not affected by storage duration but were higher when specimens were extracted using the manual method compared to the automated method (P<0.0003), regardless of cytological brush type used (Colpotre or Rovers, P<0.0022 and P<0.0445; respectively). DNA A260/280 and A260/230 ratios were comparable between the two extraction methods and collection brushes (P>0.05, all). Ninety-eight percent (53/54) of the specimens analyzed by gel electrophoresis were determined to be of acceptable integrity and the 268 bp fragment of the β-globin gene was amplified in 100% of the specimens, regardless of cytological brush type, extraction method, or storage duration.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Spectrophotometry DNA PCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Method of cell acquisition Colpotre cytological brush
Rovers Cervex-Brush-Combi cytological brush
Analyte Extraction and Purification Analyte isolation method Manual
Automated
Storage Storage duration 1-7 days
8-14 days
15-21 days
22-28 days
29-35 days
36-52 days