Activation of NF-κB signaling via cytosolic mitochondrial RNA sensing in kerotocytes with mitochondrial DNA common deletion

Scar formation as a result of corneal wound healing is a leading cause of blindness. It is a challenge to understand why scar formation is more likely to occur in the central part of the cornea as compared to the peripheral part. The purpose of this study was to unravel the underlying mechanisms. We applied RNA-seq to uncover the differences of expression profile in keratocytes in the central/peripheral part of the cornea. The relative quantity of mitochondrial RNA was measured by multiplex qPCR. The characterization of mitochondrial RNA in the cytoplasm was confirmed by immunofluoresence microscope and biochemical approach. Gene expression was analyzed by western blot and RT qPCR. We demonstrate that the occurrence of mitochondrial DNA common deletion is greater in keratocytes from the central cornea as compared to those of the peripheral part. The keratocytes with CD have elevated oxidative stress levels, which leads to the leakage of mitochondrial double-stranded RNA into the cytoplasm. The cytoplasmic mitochondrial double-stranded RNA is sensed by MDA5, which induces NF-κB activation. The NF-κB activation thereafter induces fibrosis-like extracellular matrix expressions and IL-8 mRNA transcription. These results provide a novel explanation of the different clinical outcome in different regions of the cornea during wound healing.


Results
Keratocytes with mitochondrial DNA common deletion have elevated oxidative stress and IL-8 expression. There was a higher occurrence of CD formation in the keratocytes from central cornea as compared to the limbal (i.e. peripheral) cornea (Fig. 1A). Here cells with CD were termed CD+ keratocytes, and cells without detectable CD were termed CD− keratocytes. Out of 25 samples of limbal cornea, 6 samples were CD+ , while 13 out of 20 samples of central cornea were CD+ . CD+ keratocytes showed an increased level of intracellular oxidative stress as compared to CD− keratocytes, as indicated by a ~ 2.0 fold difference in mean fluorescence intensity observed in DCF signal (Fig. 1B). Superoxide production (mitoSOX) in mitochondria in CD+ keratocytes was ~ 1.6 fold higher in mean fluorescence intensity than in CD− keratocytes (Fig. 1B). IL-8 is known to enhance keratocyte migration and recruit neutrophils, which are two necessary steps in the corneal wound healing 33 . Oxidative stress has been reported to upregulate IL-8 gene expression 34 . As expected, IL-8 expression, as measured at mRNA level, was higher in CD+ keratocytes as compared to in CD− keratocytes. There was no significant difference of IL-8 at extracellular secretion level measured by ELISA. Administration of mitoTEMPO significantly reduced the level of IL-8 expression in CD+ keratocytes (Fig. 1C-D), as well as the oxidative level in mitochondria (Additional Data Fig. 1), suggesting a positive correlation between mitochondrial ROS and IL-8 expression.
MDA5 signaling pathway induces NF-kappaB activation in CD+ keratocytes. The RNAsequencing analysis was performed to uncover the signaling pathway that might connect oxidative stress to IL-8 upregulation. There were 4485 genes that were differently expressed in CD+ keratocytes compared to in CD− keratocytes (Additional Data Fig. 2A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed several alternated pathways that were upregulated in CD+ keratocytes as compared to in CD− keratocytes. Interestingly, several enriched pathways were related to virus infection and immune response (Additional Data Fig. 2B). As we found increased IL-8 expression in CD+ keratocytes as compared to in CD− keratocytes, the KEGG enrichment pathway indicated that IL-8 upregulation could be regulated by the cytosolic RNA sensing pathway (Additional Data Fig. 2C). As shown in the diagram, RNA sensing mediated by RIG-I/ www.nature.com/scientificreports/ MDA5 could transduce the signal to activate NF-κB, which in turn induce IL-8 up-regulation. To confirm this, we measured the phosphorylated NF-κB in CD+ keratocytes, combined with the treatment of mitoTEMO. Corresponding to the pattern of IL-8 expression, NF-κB phosphorylation was higher in CD+ keratocytes, and this was abolished by mitoTEMPO treatment ( Fig. 2A), as was the case for IL-8. DDX58 and NFKBIA mRNA expression were consistent with the RNA-seq data (Fig. 2B, Additional Data Fig. 2D), while IFIH1 mRNA expression was higher in CD+ keratocytes as compared to in CD− keratocytes, which was not detected by RNA-seq data (Fig. 2B). TPCA1, an inhibitor of NF-κB nuclear localization, completely abrogated IL-8 mRNA expression (Fig. 2C), confirming a direct role of NF-κB in IL-8 regulation [35][36][37][38] . MDA5 expression was significantly reduced after siRNA transfection, as confirmed by qPCR (Fig. 2B) and western blot (Fig. 2D) in both CD+/− keratocytes. DDX58 mRNA expression was reduced 48 h after siRNA transfection ( Fig. 2B) but the corresponding RIG-I expression was barely detectable by western blot using two antibodies (MA5-31715, Invitrogen; 3743S, Cell signaling) (data not shown). siRNA-mediated MDA5 depletion ( Fig. 2D) resulted in a marked reduction of p-NF-κB in CD+ keratocytes, which was accompanied by a downregulation of IL-8 mRNA expression (Fig. 2E). These results show that IL-8 expression in CD+ keratocytes was mediated by NF-κB via MDA5 signaling.
Mitochondria double-stranded RNA (mtdsRNA) escapes from mitochondria in CD+ keratocytes. The stimulation of NF-κB can be induced by cytosolic DNA/RNA. Many studies have shown that cytosolic DNA induces NF-κB activation [39][40][41] . We first tried to determine whether cytosolic DNA was the source to induce NF-κB activation. Cytosolic nuclear DNA signal, as measured by qPCR using reference gene β2M and GAPDH probe, was close to background level (the negative control), indicating no leakage of nuclear DNA in the cytoplasm (data not shown). The source of DNA in the cytoplasm therefore could be from mitochondria. A multiplex PCR approach was applied to measure the cytosolic mtDNA content. Reduced mtDNA copy number in mitochondria was found in CD+ cells compared to in CD− keratocytes (Fig. 3A), whereas the cytosolic mtDNA content was at the same level in CD+/− keratocytes. These results contradict the possibility that cytosolic DNA triggered the immune response in CD+ keratocytes. Recent studies found that also mtRNA can induce innate immune response 26,42 . To determine whether mtRNA triggered an immune response in CD+ keratocytes, total mtRNA and cytosolic mtRNA were quantified using multiplex qPCR. The total mtRNA in CD− keratocytes was higher than that in CD+ keratocytes (Fig. 3B). This could be due to the lower mtDNA copy number in CD+ keratocytes compared to CD− keratocytes. Contrary, a significantly higher amount of mtRNA in the cytoplasm was found in CD+ keratocytes compared to in CD− keratocytes. Administration of mitoTEMPO markedly reduced the cytosolic cotent of mtRNA in CD+ keratocytes, while it has no significant effects on cytosolic mtRNA in CD− keratocytes, nor in the whole cell fraction (Fig. 3C). Dhir et al. found that mitochondria double-stranded RNA (mtdsRNA), which escaped from mitochondria into the cytosol, was a source to trigger antiviral signaling via MDA5 signaling 26 . Alternatively, RNA polymerase III (Pol III) can use cytosolic mtDNA as template to produce single-stranded mtRNA (mtssRNA) that can induce RIG-I-mediated signaling 42 . Terminator 5′-Phosphate-Dependent Exonuclease (TE) is an exonuclease that can digest RNA that www.nature.com/scientificreports/ have a 5′ monophosphate but cannot digest RNA having a 5′-triphosphate. RNA transcribed in mitochondria lacks 5′-cap protection from TE, while Pol III-transcribed RNA has a 5′-triphosphate cap. TE treatment can thus distinguish RNA transcribed in mitochondrial from RNA transcribed by Pol III in the cytoplasm. The level of mtRNA was significantly reduced after TE digestion both in the whole-cell and in the cytosolic fraction (Fig. 3D). RNA isolated from mitochondrial fraction can be completely digested by TE treatment, resulting in no detectable signal in qPCR (data not shown). Consistently, immunostaining of dsRNA with anti-dsRNA (J2) showed that J2 foci were mostly co-localized with mitochondria in both CD+/− keratocytes (Fig. 3E). Treatment of TE before J2 staining resulted in markedly reduction of J2 foci signal in keratocytes (Additional Data Fig. 3C). These results suggest that the majority of cytosolic mtRNA is from the mitochondrial matrix in CD+ keratocytes.
Altered extracellular matrix (ECM) expressions in CD+ myofibroblasts are linked to mitochondrial ROS and NF-kappaB signaling. RNA-seq data showed that col3a1 expression was significantly up-regulated in CD+ cells (Additional Data Fig. 2A). This result prompted us to study whether there were differences in collagen expression in CD+/− keratocytes, since collagen expression and organization are vitally important for corneal wound healing and also key factors altered in scar formation. Keratocytes were transformed into myofibroblasts in an in vitro fibrosis model 43 , as identified by the expression of α-SMA (Additional Data Fig. 4). mRNA expression of COL1A1, COL5A1 and lumican were lower in CD+ myofibroblasts compared to CD− myofibroblast (Fig. 4A). Pro-col I secretion from CD+ myofibroblast was significantly reduced as compared to from CD− myofibroblast (Fig. 4b). Col III secretion and mRNA expression were higher in CD+ myofibroblasts as compared to CD− myofibroblasts (Fig. 4A, top right panel, and Fig. 4B, top right panel, respectively). ECM deposition, as measured by hydroxyproline assay, showed no significant difference between CD− and CD+ myofibroblasts (Fig. 4B). To study whether mitochondrial oxidative stress and NF-κB signaling are involved in the collagen expression patterns in CD+ myofibroblasts, mitoTEMPO or TPCA treatment was used. Both mitoTEMPO and TPCA treatment effectively reversed the mRNA expression and secretion of Col I and Col III, as well as hydroproline content ( Fig. 4C-F). These results show that mitochondrial ROS and NF-κB signaling participate in the regulation of collagen expression and ECM depostion in CD+ myofibroblasts.

Discussion
CD (common deletion) has been reported to be concentrated in the central cornea as compared to in the limbal (peripheral) region 9 . Our results confirmed that a higher occurrence of CD is seen in central corneal tissue as compared to in limbal cornea. Keratocytes in the central cornea are more exposed to UV radiation compared to limbal keratocytes 6 , which means a higher risk of CD induction. As discussed by Gendron et al. 9 , the eyelid can www.nature.com/scientificreports/ cover most of the cornea in response to different light conditions but the central part is always the most exposed region of the eye 6 . Despite the observation of CD in the corneal tissue, the functional consequences of mtDNA deletion in keratocytes have not been reported. Our results showed an increase of mitochondrial superoxide, as well as a general elevation of oxidative stress, in CD+ keratocytes. However, it is not known whether the oxidative stress originated from mitochondria could affect the corneal wound healing process. Previously we have found that IL-8 enhances keratocyte migration and neutrophil recruitment 33 , which are necessary steps during corneal wound healing. However, if the neutrophil infiltration in an injured cornea is not tightly controlled, the release of various proteases may lead to stromal degradation and ulceration, which in turn might result in corneal opacity and neovascularization 32 . It is interesting to note that ROS has been positively correlated to IL-8 expression [44][45][46] . Our results confirmed that mitochondria generated ROS upregulates IL-8 expression, at both mRNA level and in secretion, by specific mitochondrial superoxide scavenger. Approximately 90% of the cellular ROS are generated in the mitochondria through OXPHOS 47 . mtDNA CD ablates several mitochondrial genes encoding for ETC. The elevated mitoSOX signal in CD+ keratocytes is thus expected (Fig. 1B), since the deficiency of ETC leads to the increased generation of ROS 48,49 . However, the mechanism behind the overall increased oxidative stress in CD+ keratocytes needs further investigation.
Besides the regulation of NF-κB by targeting related proteins directly, ROS also induces NF-κB activation indirectly by inducing DNA/RNA release into the cytoplasm. Mitochondrial ROS accumulation has been found to promote mtDNA release and initiate inflammation 24,25 . It is possible that mtRNA also escape from mitochondria in the same manner as mtDNA. Reports have shown that cytosolic RNA, from different sources, triggers the immune response. Suspene et al. proposed that cytoplasmic mtDNA can be transcribed by pol III and trigger www.nature.com/scientificreports/ www.nature.com/scientificreports/ RIG-I mediated IFN expression 42 . They found transfected mtDNA upregulated type I interferon expression in a human myeloid cell line. Dhir et al. found that mitochondrial dsRNA that had escaped into the cytoplasm in a PNPase-dependent manner triggers antiviral signaling 26 . Our RNA-seq data indicate the potential involvement of RIG-I-like receptor signaling pathway in NF-κB signaling in CD+ keratocytes (Additional Data Fig. 2). RIG-I-like receptor signaling can either be initiated by RIG-I or MDA5 RNA sensing. RIG-I specifically binds to 5′-pppRNA and short dsRNA, while MDA5 recognizes long dsRNA. Both receptors might be responsible for the induction of IL-8 in CD+ keratocytes. Both IFIH1 and DDX58 expression were higher in CD+ keratocytes as compared to that in CD− keratocytes (Fig. 2B). However, RIG-I protein was not detectable in any of the two types of keratocytes, whereas MDA5 protein level was more abundant in CD+ keratocytes (Fig. 2D), corresponding to its elevated level of IFIH1 mRNA. Notably, knockdown of IFIH1 led to reduced IL-8 mRNA in both CD+/− keratocytes (Fig. 2E), suggesting a regulatory role of MDA5 in IL-8 expression. Taken together, we confirmed that IL-8 upregulation requires the accumulation of ROS in the mitochondria and expression of MDA5, suggesting that dsRNA sensing via MDA5 accounts for the elevated IL-8 expression in CD+ keratocytes. We found the presence of mtRNA in the cytoplasm in keratocytes. Similarly, background cytosolic dsRNA was also found in HeLa cells using J2 immunofluorescence staining 26 . Although we found quantitative differences of cytosolic mtRNA by qPCR in CD+/− keratocytes, the differences were not detectable by J2 staining. The method based on immunofluorescence staining might not be sensitive enough to detect subtle changes in cytosolic dsRNA content. The higher expression of mtRNA in the qPCR data, might also be explained by the mitochondria in CD+ keratocytes leaking more, since the RNA-seq data also showed that several genes involved in mPTP were upregulated (VDAC1, VDAC2, SLC25A4 and SLC25A5). Overall, the concomitant reduction of cytosolic mtdsRNA (Fig. 3E), IL-8 expression (Fig. 1C,D), and NF-κB phosphorylation ( Fig. 2A) suggest that cytosolic mtdsRNA could activate NF-κB signaling in a mitochondrial ROS-dependent manner. It is interesting to note that IL-8 has been reported to induce NF-κB activation in a dose-dependent manner in different cell types 59 . Presumably, a positive feedback loop of IL-8-NF-κB might prolong the inflammatory state in CD+ cells in an autocrine manner.
Mitochondrial ROS generation has been found to promote mtDNA release 24,25 . Szczesny et al. found that low-level oxidative stress can also induce mtDNA oxidation and an related inflammatory response 13 . Notably, increased expression of cytoplasmic RNA sensors, such as DDX58 and IFIH1, were found in Tfam heterozygous knockout mouse cells 60 , which increase the innate immune response. These findings indicate that mtRNA escapes from mitochondria as mtDNA does. The liberation of mtRNA from mitochondria was recently reported 26 . Interestingly, they found that the release of mtdsRNA is dependent on Polynucleotide phosphorylase (PNPase). PNPase is an exoribonuclease primarily located in mitochondria 61 . It has been shown to remove oxidatively damaged RNA with high affinity 27,28,62 . There might be more mtdsRNA escape in cells under mitochondrial oxidative stress, if the capacity of PNPase is not increased correspondingly. mtDNA mutation such as CD has been proposed as a molecular marker for photoaging of skin 63,64 and cornea 9 . Photoaging is often referred to as a premature phenotype in skin caused by repeated exposure to ultraviolet (UV) radiation. Lower expression of type I pro-collagen has been found in the fibroblasts of photoaged skin compared to naturally aged skin 65 . Similarly, markedly reduced expression of collagen I (COL1A1) and collagen V (COL5A1 and A2) was observed in keratocytes treated by chronic UVA exposure as a model of photoaging 7 . Lumican, a protein that is responsible for the regulation of collagen assembly, is also downregulated in the in vitro photoaging model of corneal stroma keratocytes 7 . Collagen III is expressed weakly in the cornea under normal physiological conditions 66 . Its expression increases greatly during wound healing or inflammation and is markedly upregulated in corneal scars 67 . The elevated collagen III expression might be due to inflammation, which should be abolished-at least in later stages of wound healing-to prevent corneal scar formation. Taken together, the expression profile of CD+ cells resembles a photoaging phenotype. Considering the significant reduction of Col I expression and upregulation of Col III in photoaged keratocytes, corneal photoaging should be considered as a potential risk factor to the outcome of corneal wound healing and scar formation.
The different expression profiles of collagens in CD+/− cells suggest the potential role of NF-κB in corneal scar formation. NF-κB signaling has been reported to regulate collagen expression patterns. NF-κB activation inhibits expression of the COL1A1 68 . Conversely, reduced NF-κB activity resulted in a significant increase in COL1A1 gene expression 69 . On the opposite, Col III expression is increased with NF-κB signaling is activated 70,71 . Our results revealed the negative regulation of collagen secretion by NF-κB and mitochondrial ROS (Fig. 4 C-F). However, there was no significant difference of the actual ECM deposition between CD− and CD+ myofibroblasts (Fig. 4 B, lower right panel), indicating there were other factors determining the total collagen synthesis in CD+ myofbibroblast. Overall, targeting mitochondrial redox status and/or preventing pathological NF-κB activation during corneal wound healing could be a potential approach to counter-act corneal scar formation.

Conclusion
It is well-known that the central part of the cornea is more likely to form a scar than peripheral parts. This is especially of concern since a central corneal scar can result in a major visual problem for the patient. To be able to prevent or reduce the incidence of scar formation in the central cornea is therefore of great clinical importance. Our findings provide a novel pathway to explain the onset of permanent corneal scarring/fibrosis post-surgery or trauma. Moreover, the obtained results could also have a profound medical impact beyond corneal tissue, as a new link between mtDNA deletions and scar formation is confirmed. The study followed the principles of the Declaration of Helsinki. Cell isolation and culture were performed as described previously 1,33,72 . Corneal samples were scraped using a sterile scalpel to remove any remaining epithelial or endothelial cells, before being washed in sterile Hanks' balanced salt solution (Invitrogen, Carlsbad, CA). The remaining stromal layer was cut into 1-2 mm 2 pieces with a scalpel and then digested with 2 mg mL −1 collagenase (Sigma, St. Louis, MO) overnight at 37 °C. The suspension was centrifuged and the pellet was cultured in DMEM/F-12 media (Gibco, Carlsbad, CA) supplemented with 2% fetal bovine serum (FBS; Gibco, Carlsbad, CA) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) and placed in a humidified incubator at 37 °C with 5% CO 2 . Media was changed every third day until the cells reached confluence.

In vitro human corneal fibrosis model. The in vitro human corneal fibrosis model was adapted from
Karamichos et al. 73 and has been successfully established in our lab 43 . Corneal fibroblasts were plated on plastic culture dishes at desired densities in DMEM/F-12 10% FBS medium and stimulated by a stable vitamin C derivative L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (VitC; Sigma-Aldrich, St. Louis, MO, USA, # A8960) at a concentration of 0.5 mM, and 0.25 ng/ml recombinant human TGF-β1 (R&D Systems, # 240-B).
Separation of cytosolic DNA. Confluent cells in 10 cm dish were harvested by centrifugation (2 min, 500 g). Cells were washed twice with ice cold PBS, before resuspended in 1 ml homogenization buffer (40 mM Tris-HCl, 25 mM NaCl, 5 mM MgCl 2 , 20 ug/ml digitonin) and incubated 5 min on ice. After the homogenization, 125 μL of equilibration buffer (400 mM Tris-HCl, 250 mM NaCl, 50 mM MgCl 2 ) was added. The homogenate was centrifuged at 1200 g at 4 °C for 3 min to pellet the nuclear fraction (NF). The supernatant was collected and centrifuged at 20,000 g at 4 °C for 10 min to pellet the mitochondrial fraction (MF) and supernatant were used as cytosolic fraction (CF). NF and CF were combined and subjected to DNA extraction and subsequent multiplex qPCR for cytosolic mtDNA quantification. DNA isolation. Total DNA was isolated by PureLink Genomic DNA kit (Invitrogen, Hilden, Germany) according to the manufacturer's protocol. For cytosolic DNA isolation, 20 μL Proteinase K and 20 μL RNase A were added to 200 μL of supernatant and incubated at 55 °C for 30 min and then heated at 95 °C for 5 min. The mixture was then cooled down at room temperature and mixed with same volume of 100% ethanol. The mixture was transferred to a PureLink Spin Column and centrifuged for 15 s at > 8000 g. The flow-though was discarded. These steps were repeated to increase the DNA concentration. Washing and elution were then performed according to the standard protocols.
Separation of cytosolic RNA. The cell homogenate was prepared using the protocol for cytosolic DNA separation. The homogenate was centrifuged at 20,000 g at 4 °C for 10 min and the cytosolic fraction (CF) were used for RNA extraction (c.f. RNA isolation) and subsequent multiplex qPCR. To test the purity of cytosolic RNA in the CF extraction, the proteins from whole cell lysate (WCL), CF and pellet fraction (PF) were blotted using GAPDH, Histone H3 and TFAM antibody. No nuclear and mitochondrial proteins were detected in CF, indicating that nuclear and mitochondria lysis did not occur, thus the CF extraction was successful. The purity can also be confirmed by ponceau S staining, as different pattern of protein band can be observed (Additional Data Fig. 3).
RNA isolation. Total RNA was isolated by RNeasy/miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For cytosolic RNA isolation, 700 μl of supernatant were mixed with 100% ethanol and transferred to an RNeasy spin column placed in a 2 ml collection tube. The column was centrifuged for 15 s at > 8000 g. The flow-through was discarded. The step was repeated to increase the RNA concentration. After washing with 500 μl of Buffer RW1, the RNA is treated with DNase I while bound to the RNeasy membrane. The DNase I is removed by a second wash with Buffer RW1. Washing with Buffer RPE and elution of RNA were then performed according to the standard protocols. Western blot. Isolation and culture of primary keratocytes were performed as previously described 1 . Samples were lysed in Radioimmunoprecipitation Assay (RIPA) lysis buffer, supplemented with protease inhibitor (Sigma, St. Louis, MO) and diluted in Laemmli buffer (Bio-Rad, Hercules, CA) supplemented with β-mercaptoethanol. After boiling the samples, equal total proteins, were loaded into each well of a pre-made gel of 12% (Mini-PROTEAN TGX, Bio-Rad, Hercules, CA) and ran at 120 V for ≈60 min. Subsequently, proteins were transferred to a polyvinylidene fluoride transfer membrane (PVDF; Santa Cruz, Dallas, TX) for 60 min at 100 V. Membranes were blocked for 60 min in room temperature before primary antibody was added and incubated at 4 °C overnight. After washing, the membranes were exposed to the secondary antibody (conjugated with horseradish peroxidase, HRP) for 60 min. After additional washing the membrane was exposed to the enhanced chemiluminescence solution (GE healthcare, Little Chalfont, UK) for 5 min in room temperature. The membranes were developed using Odyssey Fc imaging system (LI-COR, Lincoln, NE). All antibodies used are summarized in Table 2.
RT-qPCR. Isolation and culture of primary keratocytes were performed as previously described 1  Other conditions were the same. Primer sequence information is summarized in Table 4.

ROS measurement.
Cells cultured in six-well plate were labeled with DCFH-DA (5 µM) and MitoSOX (5 µM) in Serum-free DMEM/F-12 media for 30 min. Subsequently, the cells were wash twice with PBS and harvested with trypsin and resuspended in 1 ml PBS. Flow Cytometry analysis was performed on FACSLSRII analysis machine and data was analyzed and illustrated by Flowing software (http:// flowi ngsoft ware. btk. fi/).
Hydroxyproline assay. Cells were treated the same as described in ELISA assay. Supernatants were collected 2 days after treatments. A hydroxyproline assay was used to determine the free hydroxyproline content in each group. This offered further insights on incorporation of free hydroxyproline in collagen synthesis. This assay was performed on hydrolyzed samples using a hydroxyproline assay kit (Abcam, Cambridge, UK). Briefly, supernatants were mixed with concentrated NaOH (10 N) in a tightened screw-capped polypropylene vial and then heated at 120 °C for 60 min. The alkaline lysate was cooled on ice, neutralized, and centrifuged to obtain  Table 4. Primers and probes used for multiplex qPCR real-time PCR. www.nature.com/scientificreports/