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The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability.

Author(s): Ayatollahi H, Sadeghian MH, Keramati MR, Ayatollahi A, Shajiei A, Sheikhi M, Bakhshi S

Publication: Niger Med J, 2016, Vol. 57, Page 199-203

PubMed ID: 27630381 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of five different DNA extraction methods on the yield, quality, and PCR success rates using frozen and FFPE lymphoid specimens. Lymphoid tissue was obtained from the ear, nose, or throat from five patients with tonsil hypertrophy undergoing tonsillectomy.  The details of FFPE and frozen specimen processing were not detailed. For each extraction method and specimen, an equal number of 10 mm thick sections were placed in microcentrifuge tubes and deparaffinized in 56˚C xylene for 10 min and rehydrated in a graded ethanol series. The DNA was then extracted using each of the five methods. The phenol chloroform method involved proteinase K digestion for 24 h at 56˚C followed by a standard phenol-chloroform extraction. The QIAamp extraction was performed according to the manufacturer’s instructions. The proteinase K and xylol extraction method was similar to the phenol chloroform extraction with the exception that the specimens were washed in phosphate-buffered saline. The alkaline heat method involved addition of NaOH and SDS to the specimen and incubation at 100˚C for 20 min followed by phenol chloroform extraction. The alkaline heat method plus mineral oil involved the addition of a 30 min incubation in mineral oil at room temperature prior to alkaline heat. DNA was assessed by spectrophotometry; amplification of 110 bp, 268 bp, and 530 bp fragments of β-globin; and real-time PCR amplification of β-actin.

   Summary of Findings:

   Interestingly, while DNA yields were higher from frozen specimens than FFPE specimens (P=0.01), the purity as determined by A260/280 or A260/230 ratios were generally comparable between the specimen types for each extraction method. The highest purity (A260/230 and A260/280) occurred when specimens were extracted using alkaline heat. The highest yields occurred when extraction was with alkaline heat and mineral oil but all extraction methods resulted in similar electrophoretic band patterns.  PCR success rates declined with increasing amplicon size, regardless of extraction method, and average cycle threshold values for β-actin were lowest when extraction was with proteinase K and xylol.  The highest success rates for amplification of the 110 bp product occurred when extraction was with proteinase K and xylol, alkaline heat with mineral oil, or phenol chloroform (100%, all). Amplification of the 256 bp product was most successful when DNA was extracted with proteinase K and xylol or phenol chloroform (100%, both). Amplification of the 512 bp product was most successful when the extraction was with alkaline heat (80%) and the next most successful were the proteinase K and xylol method and phenol chloroform method (60%, both).

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Real-time qPCR
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Real-time qPCR Specific	Targeted nucleic acid	β-actin
   PCR Specific	Targeted nucleic acid	β-globin
   PCR Specific	Length of gene fragment	128 bp 256 bp 512 bp
   Analyte Extraction and Purification	Analyte isolation method	Phenol-chloroform QIAamp FFPE Tissue kit Alkaline heat Alkaline heat plus mineral oil extraction Proteinase K and xylol extraction
   Analyte Extraction and Purification	Protein digestion	Proteinase K Incubation at 100˚C in NaOH solution

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