Absolute quantification of residual DNA in a new extracellular matrix derived from human reticular dermis (HADM) using real-time TaqMan ® MGB-PCR

Abstract

Background:

The development of dermal scaffolds is of major interest in reconstructive surgery. Human Acellular Dermal Matrices (HADMs) provides biomechanical support and elicits new tissue formation. The use of allograft dermis is limited by its immunogenic characteristics. Our research group has focused on the use of human alloplastic glycerolized reticular dermis.

Objective:

The dermal grafts were subjected to two different decellularization protocols in parallel, in order to compare the efficacy in the elimination of residual DNA.

Methods:

It was compared the incubation of the dermis in NaOH (0.06 N) and in the standard culture medium “Dulbecco Modified Eagle Medium” (DMEM). The samples were incubated in the specific medium for 8 weeks. The newly developed real-time TaqMan^® MGB-PCR assay was applied for both the detection and absolute quantification of residual DNA.

Results:

It was observed that the level of residual DNA decreased until time T3 and remained constant until time T8. Moreover, there was no statistical difference between treatment with DMEM or NaOH 0.06 N as to the amount of residual DNA.

Conclusions:

Decellularization methods, DMEM or NaOH 0.06 N do not affect DNA recovery. The proposed approach offers an alternative method to quantify residual DNA in HADM samples.

Keywords

HADM DMEM NaOH DNA extraction Taqman PCR

Abbreviations

Abb1

HADM

human acellular dermal matrice

Abb2

DMEM

Dulbecco modified eagle medium

1. Introduction

The development of dermal scaffolds is of major interest in reconstructive surgery. Human Acellular Dermal Matrices (HADMs) are used in burns, breast, pelvic and abdominal wall reconstruction as scaffolds for tissue regeneration [1,2]. HADM provides biomechanical support and elicits new tissue formation by interacting with host cells through the modulation of cellular behavior such as adhesion, migration, proliferation and differentiation. However, the use of allograft dermis as a permanent tissue replacement is limited by its immunogenic characteristics. Our research group has focused on the use of human alloplastic glycerolized reticular dermis as a scaffold for tissue regeneration. The alloplastic reticular dermis is a good matrix scaffold made up of extracellular matrix proteins, which are relatively non-immunogenic [3] and immunogenic cellular components. Decellularized dermis is non-inflammatory and has both good cell adhesion and infiltration abilities, making it suitable for clinical applications [2,4]. Decellularization techniques have been used to remove cellular components in a variety of tissues, including cardiovascular allografts and human dermal matrix, so as to produce bio-implants for clinical application. The decellularization processes remove potentially immunogenic material and provide a biocompatible scaffold for host cellular and vascular in-growth [3]. Decellularized human donor dermis should ideally provide a structurally intact natural three-dimensional extracellular matrix. Indeed, it is critical to maintain an intact extracellular matrix and remove as many of the immunogenic cells as possible so as to enhance the ability that HADM has in re-modelling and integrating into the host tissue. There are various methods able to remove the cellular component of each tissue. The most commonly used ones include a combination of physical e.g. freezing and thawing, tilting, sonication and γ ray irradiation, chemical e.g. alkaline and acid treatments, ionic, non-ionic and zwitterionic detergents etc. and enzymatic trypsin, endo and exonucleases approaches [5]. However, they all appear to unable to provide a complete decellularization as most, if not all, extracellular matrix scaffolds do retain some DNA [6,7]. In the present study we compared the efficacy of two decellularization protocols for glycerol-preserved reticular dermis in the elimination of residual DNA. The first method involved the incubation of the dermis in NaOH at a concentration of 0.06N, as previously described in literature [8]. The second method involved incubation of the dermis in the standard culture medium “Dulbecco Modified Eagle Medium” (DMEM), the tissue was shaken for up to 8 weeks in both methods.

The specimens thus obtained were then analyzed to identify and quantify any residual DNA.

Either end-point PCR or real-time PCR can be used to compare the DNA levels in different samples. The main difference between these two methods lies in the fact that, quantification takes place at the end of the entire PCR reactions in end-point PCR, whilst real-time PCR takes measurements at the exponential phase of the PCR. Absolute quantification relies on a standard plot constructed from the known concentrations of standard templates and corresponding levels of real-time PCR data. The amount is quantified with spectrophotometer at 260 nm or with DNA fluorescent dye [9] and converted into a number of copies using the molecular weight of the RNA or DNA sequence. Actual copy numbers of the gene of interest in the samples can then be read off the standard plots. Therefore, absolute quantification would eliminate the ambiguous use of ratios. However, the current methods of producing standards for absolute quantification in real-time PCR can be technically tedious, since they often involve molecular cloning of the target sequence into vector systems and amplification in Escherichia coli. In this study, we introduced another method to quantify actual copies of starting mRNA using a real-time PCR technique that does not require molecular cloning steps to obtain the standards. This method uses double-stranded (ds) DNA purified from human cells, which are quantified spectrophotometrically. The samples were also subjected to the same conditions as the standards for each assaying routine. The standard readings were used to plot a standard plot and the actual copy numbers of the samples were then read off the plot.

2. Materials and methods

2.1. Preparation of the human reticular dermal grafts

Human reticular dermal grafts that were unfit for transplantation, were harvested from the back of 28 human cadaver donors after skin donation at the Skin Bank of the Department of General and Specialized Surgery, Città della Salute e della Scienza – C.T.O. Hospital of Turin, Italy and routinely processed in the Skin Bank’s laboratory. Reticular dermis, without basement membrane, obtained with a second harvesting after the one of the skin was used to produce the matrix.

The reticular dermal grafts were placed into sterile boxes in RPMI 1640 medium (Biowest, France) and sent to the Skin Bank laboratory, where they underwent the process of glycerolisation. Standard sterile techniques were employed for all laboratory procedures. All the reticular dermal grafts were placed into a medium made up of glycerol at a concentration of 50%, amikacin (1 mg/ml) and ampicillin (600 µg/ml) and then incubated for at least three hours at room temperature. The glycerol concentration was subsequently increased to 70% and then to 85%. The allografts were agitated gently for at least three hours at $+ 33$ °C at each step.

After glycerolisation, the dermal grafts were stored at a temperature of $+ 4$ °C at least for two weeks.

The dermal grafts were then de-glycerolised and subjected to two different decellularization protocols in parallel, in order to compare the efficacy of the two techniques.

2.2. Decellularization protocols of human reticular dermal grafts

Glycerol was removed from the grafts by sequential washing in a sterile 0.9% NaCl solution at $+ 37$ °C and the grafts were then incubated by one of two methods for the decellularization: one method used DMEM (Biowest, France), a classical medium for cell culture, the other a solution of NaOH 0.06 N (Sigma, USA), this was done at room temperature with continuous tilting in both methods, as previously described in literature [6].

The samples were incubated in the specific medium for 8 weeks and the medium was changed weekly. The grafts incubated in the NaOH medium were neutralized by incubation in HCl 0.1 N before analysis.

2.3. Tissue specimens and staining procedure

A biopsy was taken after the glycerolisation procedure as a non-treated control (T0), another biopsy was taken at every week of treatment, from week 1 (T1) to week 8 (T8) after incubation in the specific medium (NaOH or DMEM). Biopsy samples were then washed in a physiologic solution, dried and conserved at a temperature of $+ 4$ °C until DNA extraction.

2.4. DNA extraction

About 50 mg of HADMs were incubated overnight at room temperature with 500 µl of lysis buffer (100 mM NaCl, 10 mMTris HCl pH8, 1 mM EDTA pH8, 1% SDS, 2% Triton X-100). The tubes were incubated at $+ 100$ ° C for 10 min. Equal volumes of phenol-chloroform were added to the sample and the tubes were centrifuged at 12,000 rpm for 10 min. Upper aqueous phase was transferred into fresh tubes, followed by adding 1 volume of ice cold ethanol, and incubated at $- 20$ °C for 1 h and centrifuged at 12,000 rpm for 10 min at $+ 4$ °C. The DNA pellets were separated from supernatants and washed twice with 70% ethanol, re-centrifuged at 12,000 rpm for 10 mins, then dried at room temperature until the smell of ethanol had been removed from the tubes. The pellets were dissolved in 20 µl of distilled water. The DNA samples were then stored at −20 °C until assayed.

2.5. Primer design and the preparation of standards

If not otherwise mentioned, the genomic DNA was used for the standard curve. The DNA was obtained from about 1 million PBMC from the donor blood bank of the University-Hospital Città della Salute e della Scienza – Molinette, Turin, Italy. The PBMC DNA were purified in the same way as that described for the HADMs. The DNA quantification was performed by spectrophotometry. The genomic DNA was used to create the standard curve by using 100 ng as the initial concentration and then five dilutions were used as follows: 1:10 dilution series for genomic DNA. Each standard dilution was tested in triplicate to ensure repeatability.

Primers and probes were designed with primer express software version 3.0 (Applied Biosystem) and were found unique for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after “blastn” (http://www.ncbi.nlm.nih.gov/BLAST/). The primer sequences were: GAPDH specific primers (GAPDHF-5′-CCAAGGTCATCCATGACAAC-3′) (GAPDHR-5′-GTGGCAGTGATGGCATGGAC-3′) and the probe was (GAPDH-6FAM-TGGTATCGTGGAAGGA-3′ MGB). Absolute Real-time PCR data were analysed with SDS 2.4 Software (Applied Biosystem). All the analyses were based on the Ct values of the PCR products i.e. the PCR cycles where the fluorescence, measured between each cycle, exceeded a threshold determined by background fluorescence at baseline and placed in the exponential phase of the amplification curve. The baseline was set automatically by the software. The SDS software calculated the standard curve for each run, based on the Ct for the standard.

2.6. Absolute quantification Real time TaqMan^® MGB

Real-time PCR was performed in Micro Amp optical 96-well plates in an automated 7500 Real Time PCR System (Life Technologies). Different concentrations of primers and probes were tested to optimize the Real time TaqMan^® MGB PCR protocols: 900 nM, 500 nM, 200 nM for primers and 250 nM, 150 nM and 100 nM for the probes. Five µl of 1/50 diluted DNA, obtained from HADMs, were added to the 2X Applied Biosystem “ready to use” master mix (Life Technologies) and 500 mM of primers and 150 nM of probes and then run in PCR under the same conditions as that of the standards. Wells with no DNA served as no template controls (NTC). The amplification conditions used were $+ 95$ °C for 10 min, followed by 40 cycles of $+ 95$ °C for 15 s and $+ 60$ °C for 1 min. A fluorescent light emission was recorded in real time and data were analysed automatically by the ABI 7500 Real-Time System SDS software (Version 2.2) for each well. The standard curve were included on each qPCR plate along with the dermis DNA extracts so as to quantify the DNA copy number in the dermis DNA extract.

2.7. Statistical analysis

Statistical analysis was performed by the Mann Whitney t-test exact test with GraphPad Prism 5 software. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Efficacy and sensitivity

These parameters were assessed by repeated testing of serial logarithmic dilutions of the standard (covering a range of five logs from 100 ng to 10 pg). The number of copies in the standard preparation used for dilutions was determined by spectrophotometry at 260 nm. After PCR amplification, the Ct value (crossing point of the amplification curve with the pre-set threshold of fluorescence detection) of individual dilution steps was plotted against the initial copy number, leading to a typical standard curve. The amplification efficacy, defined by the standard curve slope, was generally between 3 and 4. The consistency of replicates was measured by the correlation coefficient ( $R^{2}$ ), which indicates the linearity of the Ct values plotted in the standard curves. The $R^{2}$ index was higher than 0.990. The actual sensitivity of the assay was determined by the lowest standard dilution consistently detectable in replicate reactions at a frequency of 100% and it was found to be 10 pg/reaction and was linear (quantifiable) up to 10 pg. A standard dilution of 1 pg/reaction was detected at a frequency 10% (Fig. 1(A)). The reproducibility was expressed as the coefficient of variation (CV) in the $log 10$ values of the concentration. Furthermore, intra- and inter-assay variability was evaluated over different concentrations, ranging from 10 to 10³ pg within a single run ( $n = 10$ ) or different run experiments ( $n = 10$ ) by CV value of the Ct and is reported in Table 1. The quantification of dermis residue DNA was highly reproducible for as few as 10 pg.

Fig. 1.

A (top): The sensitivity of GAPDH real time TaqMan^® MGB PCR assays for the detection of residual DNA. B (bottom): The dynamic range of GAPDH real time TaqMan^® MGB PCR assays for the detection of residual DNA.

3.2. Variability

The variability was studied by analyzing 90 (9 dermis) samples tested in duplicate in five experiments. Intra- and inter-assay variations for the Ct values showed coefficients of variation (CV) 0.88% and 1.65% respectively.

3.3. Dynamic range

The dynamic range of absolute DNA quantification by PCR real time MGB was measured by serial dilutions of the standard ranging from 10⁶ to 10⁻¹ pg/reaction. The Real Time PCR assay was able to quantify DNA from 10⁵ to 10¹ pg/reaction. ( $R^{2} = 0.999$ ) (Fig. 1(B)).

3.4. Quantification of DNA in HADMs

The ng of DNA in the HADMs tested was calculated by the slope (s) and the y-axis intercept (Y) (the y-axis intercept is the point where the standard curve intersects with the ordinate; it indicates the theoretical detection limit of the reaction by revealing the Ct expected in the presence of a single target molecule in the sample) of the corresponding standard curve and the Ct of the target amplification were used according to the following equation: $Po = Inverse log (C t - Y / s)$ , where Po is the number of ng equivalents in the PCR prior to amplification. Successful amplification and quantification of DNA was obtained in all the samples.

Figure 2 and Table 2 summarize the results obtained in residual DNA dermis determination. It was observed that the level of residual DNA decreased until time T3 and remained constant until time T8. Moreover, there was no statistical difference between treatment with DMEM or NaOH 0.06 N as to the amount of residual DNA $p = 0.7209$ (Fig. 3).

Table 1
The intra and inter-assay reproducibility of the GAPDH assay. Intra and inter assay reproducibility of the GAPDH were depicted from (10 to $10^{5}$ ng/rxn). CV indicates the coefficient of variation

DNA pg Intra-assay Ct values Inter-assay Ct values

Average SD CV% Average SD CV%

10 39.75 0.33 0.84 40.31 1.95 4.85

100 36.51 0.38 1.06 36.92 0.72 1.96

1000 32.92 0.64 1.98 32.92 0.61 1.86

10000 29.54 0.41 1.45 29.46 0.50 1.71

100000 25.53 0.01 0.04 25.67 1.01 3.92

DNA pg	Intra-assay Ct values	Inter-assay Ct values
10	39.75	0.33	0.84	40.31	1.95	4.85
100	36.51	0.38	1.06	36.92	0.72	1.96
1000	32.92	0.64	1.98	32.92	0.61	1.86
10000	29.54	0.41	1.45	29.46	0.50	1.71
100000	25.53	0.01	0.04	25.67	1.01	3.92

Fig. 2.

The quantity of ng DNA obtained from HADMs at different weeks of decellularizing treatment.

Table 2

The average ng value obtained from decellularized HADMs in the GAPDH assay

	DMEM average ng/mg	NaOH average ng/mg
T0	25.09804
T1	6.996127233	11.56333312
T2	7.275	2.342455839
T3	1.876832845	0.638731597
T4	4.347461463	1.909119083
T5	3.55212	5.713056695
T6	6.314865887	6.931600151
T7	6.018867925	5.329931973
T8	1.622932528	2.103674938

Fig. 3.

Mann Whitney test. There was no statistical difference in the amount of residual DNA from HADMs decellularized in DMEM or NaOH.

4. Discussion

We used reticular dermis without basement membrane for the production of our HADM as this layer has a constitutionally low cellularity and the fact that there is no basement membrane fosters the in vitro cell penetration into extracellular matrix [10]. We chose glycerolisation as our preservation method for the production of our HADM as it is simple, cost-effective and has the capacity to produce non-viable and antiseptic grafts without damaging the extracellular matrix [11]. The goal of a decellularization protocol is that of removing all cellular and nuclear material efficiently and minimizing any adverse effect on the composition, biological activity and mechanical integrity of the remaining extracellular matrix [5]. However, they all appear to be unable to achieve complete decellularization as most, if not all, extracellular matrix scaffolds retain some DNA [6,7]. Many of the limitations related to performing PCR amplification in a clinical laboratory have been overcome by the advent of real time PCR assays. Real Time PCR assays are described as “closed systems”, since no post-amplification manipulation of the amplicon is required. The advantages of these systems over conventional PCR include a reduced turnaround time, minimizing the potential for carry-over contamination and the possibility to scrutinize the assay performance closely; all of which go to make it a suitable tool for routine diagnosis. Real Time PCR offers significant improvements in DNA quantification because of its wide dynamic range, which can accommodate at least eight log 10 copies of nucleic acid template. This is possible because the data are chosen from the linear phase of the amplification where conditions are optimal, rather than at the end-point, where the final amount of amplicon may have been affected by inhibitors, poorly optimized reaction conditions and/or saturation by inhibitory PCR by-products and double-stranded amplicon. DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing for the use of shorter probes for hybridization-based assays. In comparison to unmodified DNA, MGB probes had a higher melting temperature (Tm) and increased specificity, especially when a mismatch was in the MGB region of the duplex. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were more stabilized than G/C rich duplexes, thereby leveling probe Tm and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures [12]. The newly developed real-time TaqMan^® MGB-PCR assay was applied for both the detection and quantification of residual DNA from decellularized HADMs. The dynamic range and sensitivity of the DNA quantification assay were first evaluated by a purified DNA obtained from the PBMC of a healthy donor. TaqMan^® MGB-DNA assay showed excellent linearity between the log of target input and the CT value, demonstrating that the assay has a dynamic range of at least 7 logs able to detect as few as 1 pg in the PCR reaction (Fig. 1(A)). The sensitivity, defined as the lowest amount of target DNA detected by the assay, was determined at the concentration where the assay was positive in all replicates and was attested to 10 pg/reaction. Intra- and inter-assay variations for the Ct values showed coefficients of variation (CV) 0.88% and 1.65% respectively (Table 1). DNA quantification methods have changed substantially over the last 50 years, as investigators have exploited the complexity of the DNA molecule to develop new methods to determine the amount of DNA in a sample. Fluorescence spectroscopy provides a means to quantify samples that have even very small amounts of DNA. However, the analysis of DNA via fluorescence does necessitate the addition of an appropriate dye that will interact with the DNA. Many fluorescent dyes have been used to quantify DNA and as more dyes have become available, greater sensitivity and selectivity in the quantification of DNA have been obtained. A new generation of fluorescent DNA dyes e.g. PicoGreen, OliGreen, thiazole orange homodimer (TOTO) and oxazole yellow homodimer, have been introduced; these allow for the detection of DNA down to the picogram level [13,14]. PicoGreen can be used to detect concentrations of double-stranded DNA down to 25 pg/mL. The PicoGreen assay is linear for DNA concentrations over four orders of magnitude (from 25 pg/mL to one microgram of DNA/mL). Most of the research on scaffold biologic materials has been reported to have used PicoGreen assay for the evaluation of residual post decellularization DNA [15]. DNA quantification by real time PCR has been described in a forensic sample [16]. The advantages of the real-time assay over the plate reader assay include: a reduced hands-on time, lower assay costs and a larger dynamic range. Indeed, in this study absolute quantification with PCR standards in a real-time PCR protocol gave better accuracy and dynamic range. Real time Taqman MGB-PCR eliminates the need to incorporate molecular cloning steps into the existing real-time PCR protocol to quantify residual DNA in acellular dermis preparation. Decellularization methods, DMEM or NaOH 0.06 N do not affect DNA recovery. The proposed approach offers an alternative method to quantify residual DNA in HADM samples. We are currently improving on the approach, which is expected to increase the utility of this method. Although we shall also make efforts to determine the fragment size of the residual DNA, it is typically present as small fragments and, therefore, it is highly unlikely that they play a causative role in any adverse tissue remodeling response.

Footnotes

Acknowledgements

The work was supported by funds from the Compagnia di San Paolo, Turin, Italy, from Fondazione CRT, Turin, Italy and from Piedmont Foundation of Studies and Research on Burns Simone Teich Alasia. The authors would like to thank Barbara Wade for her linguistic advice.

Conflict of interest

The authors declare no financial or commercial conflict of interest.

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Absolute quantification of residual DNA in a new extracellular matrix derived from human reticular dermis (HADM) using real-time TaqMan ® MGB-PCR

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

Abbreviations

Abb1

Abb2

1. Introduction

2. Materials and methods

2.1. Preparation of the human reticular dermal grafts

2.2. Decellularization protocols of human reticular dermal grafts

2.3. Tissue specimens and staining procedure

2.4. DNA extraction

2.5. Primer design and the preparation of standards

2.6. Absolute quantification Real time TaqMan® MGB

2.7. Statistical analysis

3. Results

3.1. Efficacy and sensitivity

3.3. Dynamic range

3.4. Quantification of DNA in HADMs

Footnotes

Acknowledgements

Conflict of interest

References

2.6. Absolute quantification Real time TaqMan^® MGB