Table 1.
List of the different kits for the preparation of bisulfite-converted DNA from various sources.
Figure 1.
Analytical Performance of the CFF qPCR Assay.
Quantitative real-time PCR analysis of a dilution series of genomic (unconverted) and bisulfite-converted DNA (10, 5, 2.5, 1.25, 0.625 ng per PCR reaction) using the CFF assay. The CFF amplicon is free of cytosines within the sense strand and therefore allows for the amplification of bisulfite-converted and genomic DNA. Shown are mean values (± standard deviation) of triplicate measurements. The assay showed a PCR efficiency of 2.0 for both templates.
Figure 2.
DNA Yield and Purity after Bisulfite Conversion Applying Nine Different Kits.
HMW (upper panel) and FFPE tissue (lower panel) DNA was bisulfite-converted using nine different commercially available kits. Each bisulfite conversion was conducted in nine replicates for each kit. Left panel: UV spectra of bisulfite-converted and purified DNA (left panel). Middle and right panel: DNA yield of bisulfite-converted DNA as determined via UV (middle panel) measurement and via CFF qPCR assay (right panel), respectively. Shown are mean values of triplicate PCR measurements.
Table 2.
Overview over the kit performance results.
Figure 3.
Analytical Performance of the ACTB/SHOX2/SEPT9 Triplex Assay.
Bisulfite-specific and methylation-specific quantitative real-time PCR analysis of mixtures of bisulfite-converted artificially methylated DNA and unmethylated DNA from sperm. The ACTB/SHOX2/SEPT9 assay amplifies bisulfite-converted methylated SHOX2 and SEPT9 gene copies and total bisulfite-converted DNA using an ACTB amplicon. Shown are mean values (± standard deviation) of triplicate measurements.
Figure 4.
Kit Performance Comparison Using a Triplex qPCR (ACTB/SHOX2/SEPT9) Assay.
HMW (upper panel) and FFPE tissue (lower panel) DNA was bisulfite-converted using nine different commercially available kits. Each bisulfite-converted DNA was assayed using the ACTB/SHOX2/SEPT9 triplex qPCR asay. Shown are mean (± standard deviation) CT values of triplicate measurements. Left panel: ACTB CTs; middle panel: SEPT9 CTs, right panel: SHOX2 CTs.
Figure 5.
DNA Integrity after Bisulfite Conversion.
Agarose gel electrophoresis of genomic and bisulfite-converted DNA (2 μg each). The bisulfite conversion was carried out using nine different kits. Shown is bisulfite-converted DNA from HMW (left) and FFPE tissue (right) input DNA. Each bisulfite-converted DNA represents a DNA pool from nine independent bisulfite reactions per kit.
Figure 6.
PCR Inhibition and Storage Stability.
A: Inhibitory effect of eluate derived from different bisulfite conversion kits. Water was applied to nine different bisulfite conversion kits and processed like sample DNAs (process negative control sample). Different volumes (0–10μl) of this eluate were spiked into the ACTB/SEPT9/SHOX2 q PCR (20 μl total PCR volume). Each PCR contained 10 ng bisulfite-converted methylated template DNA. B, C: Storage stability. Bisulfite-converted DNA was prepared from HMW (B) and FFPE tissue (C) input DNA using nine different bisulfite conversion kits. The bisulfite-converted DNA was stored for one month at −80°C, −20°C, 4°C, and 37°C, respectively. Total amount of intact (PCR-amplifiable) DNA was determined using the CFF qPCR assay. Each kit was tested in nine replicates. Each PCR was run in duplicate. Shown are mean values (± standard deviation).
Figure 7.
Principle of the CFP Clone Sequencing Assay.
A: Basic principle of the CFP clone sequencing assay for quantifying the bisulfite conversion efficiency of the bisulfite kits. The two conversion-unspecific oligonucleotides bind to genomic sites which do not contain any cytosines and therefore amplify converted, partly converted, and unconverted DNA.
Figure 8.
Conversion Efficiency of Different Bisulfite Conversion Kits.
DNA was bisulfite converted using nine different commercially available kits. Each kit was processed in nine independent bisulfite reactions using 2 μg HMW input DNA, each. The specific conversion of cytosines to uracils at two different genetic loci were analyzed by means of the CFP clone sequencing assay.
Figure 9.
Inappropriate Conversion of Methylated Cytosines to Thymines.
A: A series of dilutions of T11 in T5MeCT5 was analyzed in the reversed-phase HPLC to determine the detection and quantification limits of the HPLC at low concentrations of T11. The percentage of T11 in the mixture of T11 and T5MeCT5 was determined by calculating ratio of the respective peak areas (area T11/(area T11+ area T5MeCT5)) B: The inappropriate conversion of methyl-cytosine to thymine was determined for nine different commercially available bisulfite conversion kits. Each bisulfite reaction was performed in triplicate.
Figure 10.
Direct Bisulfite Conversion of FFPE Tissue Sections without Prior Extraction.
Total yield of bisulfite-converted DNA derived from the direct processing of 10 μm FFPE placental tissue sections using three bisulfite conversion kits. Each kit was tested in 9 replicates. Shown are mean values (± standard deviation) of triplicate CFF qPCR measurements (B) and results from a single UV measurement (A), respectively.
Figure 11.
Direct Input of Samples Without Prior Extraction.
Yield of bisulfite-converted DNA obtained from fresh (homogenized) placental tissue (0.1 mg and 1 mg input, lower panel) and cultured cells (105 cells, upper panel) as determined by UV measurement (left panel), CFF qPCR (middle panel) and ACTB/SEPT9/SHOX2 triplex qPCR assay (right panel), respectively. Three different commercially available kits were tested in triplicate. The EZ DNA Methylation-Direct kit only allowed for the input of max. 0.1 mg tissue according to the manual. Shown are mean values (± standard deviation) of triplicate qPCR measurements and single UV determinations, respectively.
Figure 12.
Bisulfite DNA from High Volume Body Fluids.
Yield of bisulfite-converted cell-free DNA from various body fluids (urine, blood plasma and serum, ascites, and pleural effusions) as prepared using the innuCONVERT Bisulfite Body Fluids Kit. Bisulfite conversion of each sample type was done in three independent reactions. PCR measurements of each bisulfite reaction were carried in triplicate. A: Total DNA yield as quantified by the CFF qPCR assay. Shown are mean (± standard deviation) values of triplicate measurements. B: Amplification plots of the methylation-specific ACTB/SHOX2/SEPT9 qPCR.