Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine.

Author(s): Holmes EE, Jung M, Meller S, Leisse A, Sailer V, Zech J, Mengdehl M, Garbe LA, Uhl B, Kristiansen G, Dietrich D

Publication: PLoS One, 2014, Vol. 9, Page e93933

PubMed ID: 24699908 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated how bisulfate conversion kit choice may affect the yield, degradation, efficiency, inappropriate conversion rate, and stability of bisulfate-converted DNA extracted from fresh and formalin-fixed paraffin-embedded (FFPE) placenta specimens. Differences associated with obtaining DNA directly from FFPE sections or homogenized tissues using bisulfate conversion kits was also examined. DNA was extracted from five 10 µm thick FFPE sections and fresh placental tissues by overnight proteinase K digestion at 60˚C followed by phenol-chloroform extraction. Following DNA extraction, RNA was digested with RNAse I. Specimens were bisulfate-converted with each of the kits (EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit, EpiTect Fast FFPE Bisulfite Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Lightning Kit, innuCONVERT), quantified by spectrophotometry, and analyzed by real-time PCR amplification of methylation-dependent amplicons of CFF, SHOX2, and SEPT9, which were each normalized to beta actin.

   Summary of Findings:

   When high molecular weight DNA from fresh placenta specimens was used, the innuCONVERT Bisulfite and EZ DNA Methylation-Gold Kits generated the highest yields as determined by spectrophotometry, while the EpiTect Bisulfite kit generated the highest yield when quantified by the CFF PCR assay. Similarly when FFPE specimens were used, the innuCONVERT Bisulfite kits generated the highest yield of high molecular weight DNA as determined by spectrophotometry, while the EpiTect Bisulfite, innuCONVERT Bisulfite, and EZ DNA Methylation-Gold kits generated the highest yields when quantified by the CFF PCR assay. In fresh specimens, the lowest CT values for the methylation-specific amplicons of SEPT9 and SHOX2 were achieved with EZ DNA Methylation-Gold kit, followed by the innuCONVERT Bisulfite kit. In FFPE specimens, differences between kits were less pronounced but the lowest CTs for the methylation-specific amplicons of SEPT9 and SHOX2 were achieved with the EpiTect Bisulfite and the innuCONVERT Bisulfite kits. Regardless of the conversion kit used, bisulfite conversion resulted in DNA degradation and occurred to a greater extent with kits with longer durations of bisulfate conversion (EZ DNA Methylation-Gold, EZ DNA Methylation-Direct and EpiTect Bisulfite kits) compared to those that utilized a rapid protocol (EpiTect Fast FFPE, EpiTect Fast DNA Bisulfite, and the InnuCONVERT Bisulfite kits). Differences in DNA degradation among kits were less pronounced for FFPE tissues, given the degraded nature of the DNA isolated for them in comparison to fresh specimens. The efficiency of bisulfate conversion in FFPE specimens was >98.7% for all kits, with the highest rates obtained with the EZ DNA Methylation direct kits and the lowest with the EpiTect Bisulfite kit. The inappropriate conversion rate ranged from 0.9% with the innuCONVERT kits to 2.7% with the EZ DNA Methylation-Gold kit. When DNA extraction performed in tandem with bisulfate conversion using sections of FFPE or fresh tissue specimens, the innuCONVERT kit generated higher yields and amplifiability than the EZ DNA Methylation-Direct or EpiTect Fast FFPE kits. DNA prepared using innuCONVERT kit or EpiTect Fast FFPE kits from fresh tissue also performed well in downstream assays. Storage of bisulfate-converted DNA for one month at -80, -20 or 4˚C had little to no impact on the amount of amplifiable DNA via the CFF assay, regardless of the bisulfate conversion kit used or specimen type. In contrast, storage of bisulfate-converted DNA from fresh specimens for one month at 37˚C resulted in loss of the majority of amplifiable DNA via the CFF assay, regardless of bisulfate conversion kit used, but the reduction was comparably smaller among samples converted with the innuCONVERT Bisulfite kit than other kits.

   Biospecimens

   • Tissue - Placenta
   • Bodily Fluid - Ascites

   Preservative Types

   • None (Fresh)
   • Formalin

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   DNA	Bisulfite conversion assay
   DNA	Electrophoresis
   DNA	Real-time qPCR
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Formalin (buffered)
   Analyte Extraction and Purification	Analyte isolation method	innuCONVERT kit EZ DNA Methylation-Direct kit EpiTect Fast FFPE kit
   Real-time qPCR Specific	Targeted nucleic acid	CFF SEPT9 SHOX2
   Storage	Storage temperature	-80˚C -20˚C -4˚C 37˚C
   Spectrophotometry Specific	Technology platform	Real-time PCR
   Bisulfite conversion assay Specific	Technology platform	EpiTect Bisulfite Kit EpiTect Fast DNA Bisulfite Kit EpiTect Fast FFPE Bisulfite Kit EZ DNA Methylation-Direct Kit EZ DNA Methylation-Gold Kit EZ DNA Methylation-Lightning Kit innuCONVERT Bisulfite Basic Kit innuCONVERT Bisulfite All-In-One Kit

   View more

2. Study Purpose

   This study investigated potential differences in yields of bisulfate-converted cell-free DNA, and ACTB and methylation-specific amplicons (SHOX2 and SEPT9) among different specimen types. Serum, plasma, urine, pleural effusion, and ascites specimens were pooled from five to 23 cancer patients. Blood was collected in S-Monovette tubes with EDTA and Z-gel tubes for plasma and serum, respectively, then centrifuged at 1350 x g for 12 min and then again at 3000 x g for 12 min. Supernatants of pleural effusions, urine, and ascites were obtained by centrifugation at 4000 x g for 10 min. Specimens were used directly with the innuCONVERT Bisulfite Body Fluids Kit. Yield of bisulfate-converted DNA was determined by real-time PCR amplifications of CFF which amplifies bisulfate converted DNA and genomic DNA. Additionally, yield was determined by real-time PCR amplification of methylation-specific gene regions of SHOX2, and SEPT9.

   Summary of Findings:

   Yields of bisulfate-converted cell-free DNA (as determined by the CFF assay) were highest from serum specimens (297 ng/mL) followed by ascites (173 ng/mL), plasma (160 ng/mL), and pleural effusions (142 ng/mL) and were much lower from urine (21 ng/mL). Interestingly, amplification of a methylation-specific amplicon of SEPT9 was unsuccessful in cell-free DNA isolated from ascites or pleural effusions, but was possible with DNA isolated from urine and was abundant in plasma and serum specimens. While generation of a methylation-specific amplicon of SHOX2 was observed after less than 35 cycles using DNA isolated from plasma and serum, a similar amplicon intensity was not observed until more than 45 cycles when DNA isolated from urine, ascites, or pleural effusion specimens was used. 

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Serum
   • Bodily Fluid - Urine
   • Bodily Fluid - Peritoneal Fluid
   • Bodily Fluid - Pleural Fluid
   • Bodily Fluid - Ascites

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Neoplastic - Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Bisulfite conversion assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qPCR Specific	Targeted nucleic acid	CFF SEPT9 SHOX2
   Biospecimen Acquisition	Biospecimen location	Plasma Serum Urine Ascites Pleural effusions
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum

   View more