With numerous kits on the market for PCR of methylated DNA, researchers may wonder which is the best for bisulfite conversion of various types of real-world samples. A study published this week in PLoS One compared nine kits from three different companies – Qiagen, Zymo Research, and Analytik Jena – and found Analytik Jena's bisulfite conversion kits had higher DNA yield from formalin-fixed, paraffin-embed tissues without prior extraction, and were more versatile in terms of sample material.
The nine kits assayed included Qiagen's EpiTect Bisulfite, Fast DNA Bisulfite, and Fast FFPE Bisulfite kits; Zymo's EZ DNA Methylation-Direct, Methylation-Gold, and Methylation-Lightning kits; and Analytik Jena's innuConvert Bisulfite Basic, Bisulfite All-In-One, and Bisulfite Body Fluids kits.
In an interview with PCR Insider, Dimo Dietrich, a researcher at Bonn University Hospital in Germany and an author of the study, said he was motivated to publish this comparison by a perceived need in the field. Since clinical samples can be quite varied, Dietrich's team evaluated the kits using DNA obtained from fresh tissues, as well as from FFPE and large volumes of body fluids. "I realized that researchers need to be able to analyze very specific clinical specimens, and usually companies optimize their kits with regard to sample material which is easily available, like extracted DNA or cell line tissues," he said.
The upshot of the study was that "they are all pretty good" for the intended use of each kit, Dietrich said.
However, products from Analytik Jena's innuConvert line were better in some regards. "The main advantage is that the innuConvert product is applicable to many more different types of specimens, and that it's much faster," said Dietrich. The innuConvert Bisulfite Body Fluids kit, for example, is "the only product which is applicable to all kinds of samples with clinical relevance, like plasma, effusions, lavages, and so on," Dietrich said.
Although Dietrich serves as a scientific consultant to Analytik Jena, the head-to-head assessment was based on his desire to find the ideal tools for his own research. "I have ten years experience in developing bisulfite methods, and also pre-analytical steps ... I'm also very much into the paraffin-embedded tissue, and a lot of the methodological background comes from my expertise."
"I think we did a very fair comparison, we stuck to the protocols recommended by all companies," he added. "For example, we found that this one product from Zymo research failed when you analyzed paraffin-embedded tissue sections, and this is most likely because they simply don’t use enough proteinase K, as you can see at first glance when you look into their protocol. But we did not change anything in the protocols, to be sure that it's a fair comparison."
Dietrich and his group first prepared stocks of pooled DNA, then ran nine replicates for each kit, assay, and type of specimen. According to the study, "kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis."
The study compared DNA from fresh and FFPE placental tissue, as well as plasma, serum, urine, plural effusions, and ascites, where appropriate. For FFPE, they compared extracted DNA, as well as direct conversion without prior extraction. All this was controlled with unmethylated and methylated reference DNA.
For FFPE without prior extraction, the innuConvert All-In-One kit was able to obtain substantially higher yields of DNA, as measured by UV and qPCR for a cytosine-free fragment, or CFF assay.
In addition, with this Analytik Jena kit line, "the conditions are less harsh, and the integrity of the DNA is higher, so the amplification of larger amplicons is more robust," Dietrich said.
The study used a triplex qPCR of the converted DNA, measuring two cancer-associated genes and a reference gene. Dietrich believes this assay provides a fair assessment of the functionality of each kit. "The triplex contains two cancer biomarkers, SHOX2 and SEPTIN9, and actin beta as a housekeeping gene in which the PCR amplicon does not cover any CpG sites. So it should work in all kits," he said. The group also ran the CFF qPCR, "so all together we had almost four independent PCRs." Dietrich said, "We saw that higher variability goes hand in hand with higher fragmentation, so the overall picture makes sense. I don't expect any surprises when you use other downstream assays for analysis."
The study also calculated hands-on time and time-to-results for each kit. "Hands-on time is the time you really need to be in the lab." Dietrich said. "For example the EpiTect bisulfite kit has hands-on time of only 97 minutes, even though time-to-result is 402 minutes. This is simply because you have a five-hour incubation step in between. Hands-on time is more important when you calculate the cost for labor, and time to results is important when you need quick results," he said.
The Analytik Jena kit for body fluids is the only kit for bisulfite conversion of large volumes of fluids currently on the market, according to Dietrich. This product uses polymer-mediated extraction, which is also used in Analytik Jena's cell-free DNA sample prep kit, launched earlier this year.
Analytik Jena acquired California-based UVP one year ago as part of its effort to expand its foothold in the US market. AJ's products are available through UVP, and a representative at that company said they are also distributed via VWR.
The kits used in the study range in cost. The three from Zymo are between $134 and $178, while those from Qiagen are $195 to $346. A representative of Analytik Jena told PCR Insider in an email that their "products have been priced at a competitive price-performance ratio."
A Qiagen spokesperson emphasized that, in the study, all three kits were comparable in many regards, and that Qiagen's kit has some advantages.
Its "proprietary DNA-protect buffer helps to minimize DNA damage during sample processing while its pH indicator, which turns from green to blue, serves as an easy-to-follow, in-process control for proper mixing and correct pH of the reaction," the spokesperson wrote in an email to PCR Insider.
"This makes the EpiTect Fast kits particularly suitable for FFPE samples where the starting material is already substantially fragmented," the spokesperson added. In addition, "using Qiagen's benchtop-sized automation platform QIAcube for the purification protocol, the total hands-on time of all available EpiTect and EpiTect Fast kits can be reduced to far below one hour." In addition, the QIAamp Circulating Nucleic Acid can be used with up to 5 ml of serum or plasma or other cell-free body fluids to collect and purify cell-free DNA, and the "minimal elution volume of 20 µl fits perfectly to feed into the EpiTect Fast bisulfite conversion protocol," according to the company.
Qiagen also asserted that the study shows that "among the three tested kits that allow for combined cell lysis and bisulfite conversion, EpiTect Fast is the best match for bisulfite conversion efficiency, DNA purity and DNA integrity, and fastest time to result."
Zymo did not respond to a request for comment in time for publication.
For the purposes of the kit comparisons, Dietrich's group adhered strictly to the protocols provided with each kit, but Dietrich was also the architect of a recently published method to enhance PCR from FFPE, as covered in PCR Insider. However, the lab now routinely uses these simple fixes involving increasing reagent concentrations in other studies on FFPE. "It's really helpful," Dietrich said. "I already got some feedback from two other researchers who confirmed these results, and mentioned that they now have better results."
Dietrich is currently assessing the potential of SHOX2 and SEPTIN9 as plasma biomarkers in about 100 patients followed after surgeries to treat head and neck cancers. The kit comparison will enable that research. "We have all our tools which that we need to measure clinical samples. Now I want to see if I can detect any relapse early so that [patients] can get better therapy," he said.