Notice & News
Circulating cell-free DNA (cfDNA) is gaining prevalence as a non-invasive, alternative approach for the detection of tumor mutations in cancer management (1) and screening tests for fetal abnormalities from the mother’s blood (2). cfDNA is known to circulate in healthy and pathological conditions and is present in plasma, serum, cerebral spinal fluid, and saliva. Evidence has suggested that highly fragmented cfDNA is actively secreted when new nucleic acids are synthesized and passively released as an end product of necrosis and apoptotic cell death (1).
The increased use of cfDNA has prompted interest in the best evaluation method for cfDNA. In this application note, we compared cfDNA peak separation using two reagent kits and two different capillary arrays for the Fragment Analyzer™ Automated CE System: DNF-477, High Sensitivity Small Fragment Analysis Kit and DNF-464, High Sensitivity Large Fragment 50 Kb Analysis Kit with the short and ultra-short arrays. As the extraction methods play a key role in quantity and quality of cfDNA recovery, we also compared the cfDNA separation profile on the Fragment Analyzer after extraction with two different kits.
The Fragment Analyzer, equipped with the short (33 cm effective, 55 cm total) or ultra-short (22 cm effective, 47 cm total) array was used to analyze a 1.25 ng/µL or 2.5 ng/µL cfDNA sample with DNF-477, High Sensitivity Small Fragment Analysis Kit (HS Small Fragment Kit) and DNF-464, High Sensitivity Large Fragment 50 Kb Analysis Kit (HS Large Fragment Kit). cfDNA was extracted from four healthy human serum samples (#HMSRM) obtained from Bioreclamation IVT using the QIAmp ® Circulating Nucleic Acid Kit (Qiagen #55114) or the Quick-cfDNA™ Serum & Plasma Kit (Zymo #D4076). While carrier RNA is not used in the Quick-cfDNA Kit, the QIAmp Kit utilized carrier RNA for the extraction of cfDNA. All serum samples extracted with the QIAmp Kit were split into two 5 mL samples and extracted with and without carrier RNA to address possible carrier RNA interference with cfDNA peak separation. Some cfDNA samples were also treated with DNase to further investigate possible peak interference. Qubit ® 2.0 was used for quantification of cfDNA.
Results & Discussion
Separation of cfDNA by HS Small Fragment Kit with Short Array
The HS Small Fragment Kit distinctively separated three cfDNA peaks from healthy human serum at 157 bp, 349 bp, and 559 bp (Figure 1A, red trace). These peak sizes corresponded to a nucleosome guided fragmentation pattern of apoptotic cfDNA, oftentimes referred to as mono-nucleosome, di-nucleosome, and tri-nucleosome cfDNA [3,4]. The tri-nucleosome peak is not always seen and may be overshadowed by carrier RNA used in some kits. To investigate the possible interference of carrier RNA with the tri-nucleosome peak, DNase (Figure 1A, black trace) was added to cfDNA samples extracted with carrier RNA by the QIAmp kit. Figure 1A showed that DNase completely degraded everything in the cfDNA sample extracted with carrier RNA on the HS Small Fragment Kit. The third peak detected is indeed cfDNA and not carrier RNA (Figure 1A, red trace). This was confirmed by detection of three peaks with similar base pair lengths from cfDNA extracted by the Quick-cfDNA Kit, which does not utilize carrier RNA (Figure 1B).
Figure 1 - cfDNA separated using the HS Small Fragment Kit on the Fragment Analyzer™ short array. (A) cfDNA extracted with the QIAmp Kit using carrier RNA and treated with DNase (black trace) and without DNase (red trace). (B) cfDNA extracted without carrier RNA using the Quick-cfDNA Kit.
Separation of cfDNA by HS Large Fragment Kit with Short Array
cfDNA from Sample 1 was also run using the HS Large Fragment Kit and three peaks at 162 bp, 347 bp, and 551 bp were separated (Figure 2A, red trace). The possible interference of carrier RNA was explored by the addition of DNase to this cfDNA sample extracted with carrier RNA. In Figure 2A, the DNase treated cfDNA (black trace) displayed a distinct smear averaging 487 bp. Since DNase degrades only DNA, the remaining smear was likely carrier RNA. The carrier RNA smear stretched from 450 to 700 bp, overlapping with the 551 cfDNA base pair peak. These results clearly showed that carrier RNA interfered with the third cfDNA peak separation on the HS Large Fragment Kit. cfDNA extracted without carrier RNA by the Quick-cfDNA Kit likewise separated three cfDNA peaks on the HS Large Fragment Kit (Figure 2B). The Fragment Analyzer detected three cfDNA peaks on both the HS Small Fragment Kit and the HS Large Fragment Kit in the cfDNA samples tested. Similar separation profiles were observed for samples #1, #2, and #3, sample #1 is shown in Figures 1 and 2.
Figure 2 - cfDNA separated using the HS Large Fragment Kit on the Fragment Analyzer™ short array. Graphs were expanded and the upper marker cut off for comparison. (A) cfDNA extracted using the QIAmp Kit with carrier RNA and treated with DNase (black trace) and without DNase (red trace). (B) cfDNA extracted without carrier RNA with the Quick-cfDNA Kit.
Separation of cfDNA Containing gDNA
To evaluate if carrier RNA separates along with the fragmented genomic DNA, Sample #4 was extracted with carrier RNA (black trace) and without carrier RNA (red trace). The results indicate that carrier RNA did not affect the profile of cfDNA when separated by the HS Small Fragment Kit (Figure 3A). Sample #4 was also separated with the HS Large Fragment Kit. The HS Large Fragment Kit was capable of completely separating cfDNA with fragmented genomic DNA (Figure 3B). The genomic DNA smear occurred from 760 bp to 6,000 bp. However, cfDNA extracted with carrier RNA (black trace) continued to interfere with the separation of the 582 bp cfDNA peak on the HS Large Fragment Kit.
The separation profile of cfDNA from sample #4 using the HS Small Fragment Kit is shown in Figure 3A. In addition to the three standard nucleosome peaks, sample #4 contained fragmented genomic DNA.The smear started at 800 bp and ran through the 1,500 bp upper marker (Figure 3A). An extended run time was utilized to complete the DNA separation with the HS Small Fragment Kit.
Figure 3 - cfDNA sample #4 containing fragmented genomic DNA was separated on the Fragment Analyzer™ short array. (A) cfDNA extracted with carrier RNA (black trace) and without carrier RNA (red trace) on the HS Small Fragment Kit. (B) cfDNA extracted with carrier RNA (black trace) and without carrier RNA (red trace) on the HS Large Fragment Kit.
Comparison of Different cfDNA Extraction Kits
The concentration of cfDNA found in serum samples is extremely low. Therefore utilizing the most effective extraction kit is a necessity.We compared the total cfDNA extraction yield from 5 mL of healthy human serum between the QIAmp Kit and the Quick-cfDNA Kit (Table 1).
Table 1: Comparison of the total yield from the QIAmp Kit and the Quick-cfDNA Kit.
a N=4 from same sample.
Comparison Between Short Array and Ultra-Short Array
The Fragment Analyzer ultra-short array was utilized to reduce analysis time of cfDNA extracted by the Quick-cfDNA Kit. The separation time was reduced by 10 minutes for the HS Small Fragment Kit (Figure 4A) and by 15 minutes for the HS Large Fragment Kit (Figure 4B). Both kits produced excellent separations of the cfDNA peaks on the Fragment Analyzer ultra-short array.
Figure 4 - cfDNA extracted by Quick-cfDNA Kit was separated on the Fragment Analyzer™ ultra-short array. (A) HS Small Fragment Kit. (B) HS Large Fragment Kit.
Table 2 summarizes the differences between the HS Small Fragment Kit and HS Large Fragment Kit for cfDNA analysis. The HS Small Fragment Kit provided efficient and excellent separations of cfDNA extracted with or without carrier RNA, however it could not completely separate fragmented genomic DNA. The HS Large Fragment Kit completely separated cfDNA that contained fragmented genomic DNA, but displayed interference from carrier RNA. When possible, cfDNA extracted without carrier RNA is recommended to avoid carrier RNA interference.