Progressive Thinning of Retinal Nerve Fiber Layer/Ganglion Cell Layer (RNFL/GCL) as Biomarker and Pharmacological Target of Diabetic Retinopathy.

Diabetes-driven retinal neurodegeneration has recently been shown to be involved in the initial phases of diabetic retinopathy, raising the possibility of setting up a preventive strategy based on early retinal neuroprotection. To make this possible, it is crucial to identify a biomarker for early retinal neurodegeneration. To this end, in this study, we verified and confirmed that, in the Akita mouse model of diabetes, the thinning of the retinal nerve fiber layer/ganglion cell layer (the RNFL/GCL-the layer that contains the retinal ganglion cells) precedes the death of these same cells, suggesting that this dysfunction is a possible biomarker of retinal neurodegeneration. We then confirmed the validity of this assumption by starting a neuroprotective treatment (based on nerve growth factor eye drops) in concert with the first demonstration of RNFL/GCL thinning. In this way, it was possible not only to avoid the loss of retinal ganglion cells but also to prevent the subsequent development of the microvascular stage of diabetic retinopathy. In conclusion, in the case of diabetes, the thinning of the RNFL/GCL appears to be both a valid biomarker and a pharmacological target of diabetic retinopathy; it precedes the development of vascular dysfunctions and represents the ideal starting point for prevention.

Early increase in circulating carbonic anhydrase IX: A potential new predictive biomarker of preeclampsia.

Preeclampsia (PE) is a severe complication of pregnancy. The identification of a reliable predictive biomarker could help in setting up a specific preventive strategy. To this aim, we studied carbonic anhydrase IX (CAIX) as a marker of hypoxia (a pathway involved in PE pathogenesis) and compared the diagnostic accuracy of CAIX to that of the validated biomarker sFlt1/PlGF ratio. Fifteen women with overt PE and 38 women at a risk of developing PE, sampled at different time intervals during gestation (a total of 82 plasma samples collected), were enrolled and underwent the CAIX measurement. CAIX levels significantly increased (p < .001) before the onset of the disease in women (25% of the total number) who later on developed PE when compared to women who did not, starting from 28th gestational week. The best CAIX cut-off of 68.268 pg/mL yielded a sensitivity of 100%, a specificity of 81.82%, and an AUC value of .9221. In our pilot study, when compared to the sFlt1/PlGF ratio, CAIX performed better in predicting PE before the clinical onset. Furthermore when implemented as CAIX/PlGF ratio, showed up to be comparable in the identification of women with overt early PE. In conclusion, CAIX could represent an effective predictive biomarker of PE, and larger studies are mandatory to validate this finding.

Dual-Domain Reporter Approach for Multiplex Identification of Major SARS-CoV-2 Variants of Concern in a Microarray-Based Assay.

Since the emergence of the COVID-19 pandemic in December 2019, the SARS-CoV-2 virus continues to evolve into many variants emerging around the world. To enable regular surveillance and timely adjustments in public health interventions, it is of the utmost importance to accurately monitor and track the distribution of variants as rapidly as possible. Genome sequencing is the gold standard for monitoring the evolution of the virus, but it is not cost-effective, rapid and easily accessible. We have developed a microarray-based assay that can distinguish known viral variants present in clinical samples by simultaneously detecting mutations in the Spike protein gene. In this method, the viral nucleic acid, extracted from nasopharyngeal swabs, after RT-PCR, hybridizes in solution with specific dual-domain oligonucleotide reporters. The domains complementary to the Spike protein gene sequence encompassing the mutation form hybrids in solution that are directed by the second domain ("barcode" domain) at specific locations on coated silicon chips. The method utilizes characteristic fluorescence signatures to unequivocally differentiate, in a single assay, different known SARS-CoV-2 variants. In the nasopharyngeal swabs of patients, this multiplex system was able to genotype the variants which have caused waves of infections worldwide, reported by the WHO as being of concern (VOCs), namely Alpha, Beta, Gamma, Delta and Omicron variants.

Topical nerve growth factor prevents neurodegenerative and vascular stages of diabetic retinopathy.

Specific and effective preventive treatment for diabetic retinopathy (DR) is presently unavailable, mostly because the early stages of the complication have been, until recently, poorly understood. The recent demonstration that the vascular phase of DR is preceded and possibly caused by the neurodegeneration of retinal ganglion cells suggests that DR could, at least theoretically, be prevented through an early neuroprotective approach. The aims of our study were to clarify the natural history of diabetes-driven retinal neurodegeneration and to verify the possibility to prevent DR using topical nerve growth factor (NGF). The results of the study show that retinal neurodegeneration, characterized by the loss of retinal ganglion cells represents a relatively early phenomenon of diabetes (between 5 and 16 weeks of age), which tends to be self-limiting in the long run. Neurodegeneration is followed by the development of DR-related vascular dysfunctions, as confirmed by the development of acellular capillaries and the loss of retinal pericytes. Both retinal neurodegeneration and subsequent vascular dysfunction can be successfully prevented by topical NGF administration. These findings suggest that: 1) The first stage of DR consists in a self-limiting retinal neurodegeneration 2) The demonstrated effectiveness of topical NGF in the prevention of DR could be rapidly translated into clinical practice.

Cyclosporine A Inhibits Viral Infection and Release as Well as Cytokine Production in Lung Cells by Three SARS-CoV-2 Variants.

In December 2019, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started spreading worldwide causing the coronavirus disease 2019 (COVID-19) pandemic. The hyperactivation of the immune system has been proposed to account for disease severity and death in COVID-19 patients. Despite several approaches having been tested, no therapeutic protocol has been approved. Given that Cyclosporine A (CsA) is well-known to exert a strong antiviral activity on several viral strains and an anti-inflammatory role in different organs with relevant benefits in diverse pathological contexts, we tested its effects on SARS-CoV-2 infection of lung cells. We found that treatment with CsA either before or after infection of CaLu3 cells by three SARS-CoV-2 variants: (i) reduces the expression of both viral RNA and proteins in infected cells; (ii) decreases the number of progeny virions released by infected cells; (iii) dampens the virus-triggered synthesis of cytokines (including IL-6, IL-8, IL1α and TNF-α) that are involved in cytokine storm in patients. Altogether, these data provide a rationale for CsA repositioning for the treatment of severe COVID-19 patients. IMPORTANCE SARS-CoV-2 is the most recently identified member of the betacoronavirus genus responsible for the COVID-19 pandemic. Repurposing of available drugs has been a "quick and dirty" approach to try to reduce mortality and severe symptoms in affected patients initially, and can still represent an undeniable and valuable approach to face COVID-19 as the continuous appearance and rapid diffusion of more "aggressive"/transmissible variants, capable of eluding antibody neutralization, challenges the effectiveness of some anti-SARS-CoV-2 vaccines. Here, we tested a known antiviral and anti-inflammatory drug, Cyclosporine A (CsA), and found that it dampens viral infection and cytokine release from lung cells upon exposure to three different SARS-CoV-2 variants. Knock down of the main intracellular target of CsA, Cyclophilin A, does not phenocopy the drug inhibition of viral infection. Altogether, these findings shed new light on the cellular mechanisms of SARS-CoV-2 infection and provide the rationale for CsA repositioning to treat severe COVID-19 patients.

Renal dysfunction and podocyturia in pre-eclampsia may be explained by increased urinary VEGF.

BACKGROUND: Pre-eclampsia has a major impact on renal function as shown by the development of proteinuria and podocyturia. How the systemic, soluble Fms-like tyrosine kinase-1 (sFlt-1)-driven inhibition of vascular endothelial growth factor (VEGF) activity detected in pre-eclampsia directly affects renal function remains unknown. The aim of the study was to clarify whether a non-canonical, renal-centred escape from VEGF inhibition in the case of pre-eclamptic pregnancy might have a direct impact on renal function. METHODS: We evaluated plasma and urinary VEGF and placental growth factor (PlGF), plasma sFlt-1 and carbonic anhydrase IX (CAIX), albuminuria and podocyturia in 18 women with uncomplicated pregnancy, 21 with pre-eclampsia and 18 non-pregnant. The three groups were matched for age and the pregnant groups also for gestational age at enrolment. RESULTS: Plasma VEGF was reduced in uncomplicated (P = 0.001) and pre-eclamptic (P = 0.0003) pregnancies when compared with controls. In uncomplicated pregnancy, the dysfunction was balanced by an increase (P = 0.009) of plasma PlGF. Increased (P = 0.0001) plasma CAIX in pre-eclampsia was in line with hypoxia. Pre-eclampsia resulted in a paradoxical increase (P = 0.0004) of urinary excretion of VEGF. Urinary concentrations of VEGF and podocytes were correlated to each other (r2 = 0.48, P < 0.0005) but also to plasma sFlt-1 (r2 = 0.56, P < 0.0001 and r2 = 0.23, P = 0.03, respectively). CONCLUSIONS: In the case of pre-eclampsia, the systemic VEGF inhibition leads the kidney, possibly the podocyte, to increase the VEGF synthesis. The mechanisms leading to local VEGF overproduction or the overproduced VEGF itself are reasonably involved in the pathogenesis of podocyturia and, as a consequence, renal dysfunction in pre-eclampsia.

Common pathways in dementia and diabetic retinopathy: understanding the mechanisms of diabetes-related cognitive decline.

Type 2 diabetes (T2D) is associated with multiple comorbidities, including diabetic retinopathy (DR) and cognitive decline, and T2D patients have a significantly higher risk of developing Alzheimer's disease (AD). Both DR and AD are characterized by a number of pathological mechanisms that coalesce around the neurovascular unit, including neuroinflammation and degeneration, vascular degeneration, and glial activation. Chronic hyperglycemia and insulin resistance also play a significant role, leading to activation of pathological mechanisms such as increased oxidative stress and the accumulation of advanced glycation end-products (AGEs). Understanding these common pathways and the degree to which they occur simultaneously in the brain and retina during diabetes will provide avenues to identify T2D patients at risk of cognitive decline.

SARS-CoV-2 Entry: At the Crossroads of CD147 and ACE2.

In late 2019, the betacoronavirus SARS-CoV-2 was identified as the viral agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. Coronaviruses Spike proteins are responsible for their ability to interact with host membrane receptors and different proteins have been identified as SARS-CoV-2 interactors, among which Angiotensin-converting enzyme 2 (ACE2), and Basigin2/EMMPRIN/CD147 (CD147). CD147 plays an important role in human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, Kaposi's sarcoma-associated herpesvirus, and severe acute respiratory syndrome coronavirus infections. In particular, SARS-CoV recognizes the CD147 receptor expressed on the surface of host cells by its nucleocapsid protein binding to cyclophilin A (CyPA), a ligand for CD147. However, the involvement of CD147 in SARS-CoV-2 infection is still debated. Interference with both the function (blocking antibody) and the expression (knock down) of CD147 showed that this receptor partakes in SARS-CoV-2 infection and provided additional clues on the underlying mechanism: CD147 binding to CyPA does not play a role; CD147 regulates ACE2 levels and both receptors are affected by virus infection. Altogether, these findings suggest that CD147 is involved in SARS-CoV-2 tropism and represents a possible therapeutic target to challenge COVID-19.

CovidArray: A Microarray-Based Assay with High Sensitivity for the Detection of Sars-Cov-2 in Nasopharyngeal Swabs.

A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (Covid-19) epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions, RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false negatives are reported. Moreover, RT-qPCR requires up to 3-6 h with most of the time involved in RNA extraction from swab samples. We introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid-phase hybridization of fluorescently-labeled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows the reduction of both the amount of false-negative results and the total analysis time to about 2 h.

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis.

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

Updates on liquid biopsy: current trends and future perspectives for clinical application in solid tumors.

Despite advances in screening and therapeutics cancer continues to be one of the major causes of morbidity and mortality worldwide. The molecular profile of tumor is routinely assessed by surgical or bioptic samples, however, genotyping of tissue has inherent limitations: it represents a single snapshot in time and it is subjected to spatial selection bias owing to tumor heterogeneity. Liquid biopsy has emerged as a novel, non-invasive opportunity of detecting and monitoring cancer in several body fluids instead of tumor tissue. Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), RNA (mRNA and microRNA), microvesicles, including exosomes and tumor "educated platelets" were recently identified as a source of genomic information in cancer patients which could reflect all subclones present in primary and metastatic lesions allowing sequential monitoring of disease evolution. In this review, we summarize the currently available information concerning liquid biopsy in breast cancer, colon cancer, lung cancer and melanoma. These promising issues still need to be standardized and harmonized across laboratories, before fully adopting liquid biopsy approaches into clinical practice.

Small EVs-Associated DNA as Complementary Biomarker to Circulating Tumor DNA in Plasma of Metastatic Colorectal Cancer Patients.

It is widely accepted that assessing circular tumor DNA (ctDNA) in the plasma of cancer patients is a promising practice to evaluate somatic mutations from solid tumors noninvasively. Recently, it was reported that isolation of extracellular vesicles improves the detection of mutant DNA from plasma in metastatic patients; however, no consensus on the presence of dsDNA in exosomes has been reached yet. We analyzed small extracellular vesicle (sEV)-associated DNA of eleven metastatic colorectal cancer (mCRC) patients and compared the results obtained by microarray and droplet digital PCR (ddPCR) to those reported on the ctDNA fraction. We detected the same mutations found in tissue biopsies and ctDNA in all samples but, unexpectedly, in one sample, we found a KRAS mutation that was not identified either in ctDNA or tissue biopsy. Furthermore, to assess the exact location of sEV-associated DNA (outside or inside the vesicle), we treated with DNase I sEVs isolated with three different methodologies. We found that the DNA inside the vesicles is only a small fraction of that surrounding the vesicles. Its amount seems to correlate with the total amount of circulating tumor DNA. The results obtained in our experimental setting suggest that integrating ctDNA and sEV-associated DNA in mCRC patient management could provide a complete real-time assessment of the cancer mutation status.

Membrane-binding peptides for extracellular vesicles on-chip analysis.

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.

Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour dna Mutations.

: It has now been established that in biological fluids such as blood, it is possible to detect cancer causing genomic alterations by analysing circulating tumour DNA (ctDNA). Information derived from ctDNA offers a unique opportunity to enrich our understanding of cancer biology, tumour evolution and therapeutic efficacy and resistance. Here, we propose a workflow to identify targeted mutations by a customized microarray-based assay for the simultaneous detection of single point mutations in different oncogenes (KRAS, NRAS and BRAF) followed by droplet digital PCR (ddPCR) to determine the fractional abundance of the mutated allele. Genetic variants were determined in the plasma of 20 metastatic colorectal cancer (mCRC) patients previously genotyped on tissue biopsy at the diagnosis for medication planning (T0) and following the tumour genetic evolution during treatment phase (T1 and T2) with the objective of allowing therapy response prediction and monitoring. Our preliminary results show that this combined approach is suitable for routine clinical practice. The microarray platform enables for a rapid, specific and sensitive detection of the most common mutations suitable for high-throughput analysis without costly instrumentation while, the ddPCR, consents an absolute quantification of the mutated allele in a longitudinal observational study on patients undergoing targeted therapy.

Evaluation of three advanced methodologies, COLD-PCR, microarray and ddPCR, for identifying the mutational status by liquid biopsies in metastatic colorectal cancer patients.

A major effort has been focused on the detection of oncogenes' mutations in diverse types of clinical specimens including formalin-fixed and paraffin embedded tissues, presently the gold-standard samples, up to plasma, that constitute a noninvasive alternative source of tumor DNA. The reliable detection of mutations in circulating tumor DNA requires a high analytical sensitivity. Here, we applied three different highly sensitive methodologies (COLD-PCR, a microarray-based approach and the droplet digital PCR, ddPCR) to identify mutations in the plasma of 30 metastatic colorectal cancer patients previously genotyped on tissue biopsy. The methods showed a modest concordance rate with respect to the results obtained on tissue biopsies: 63.3% by ddPCR, 63% by microarray and 55.6% by COLD-PCR. This could be ascribed either to the different timing between tissue and liquid biopsy collection, which could reflect a different stage of disease progression or to the diverse sensitivity of the methodologies applied. Indeed, if we compare the results obtained on plasma samples, the concordance rates were higher especially by comparing ddPCR versus COLD-PCR (92.6%). Thus, we consider both methodologies as useful procedures easily transferable in a clinical setting. Notably, the ddPCR allows a quantitative assessment of the fractional abundance of the mutation.

Analysis of KRAS, NRAS and BRAF mutational profile by combination of in-tube hybridization and universal tag-microarray in tumor tissue and plasma of colorectal cancer patients.

Microarray technology fails in detecting point mutations present in a small fraction of cells from heterogeneous tissue samples or in plasma in a background of wild-type cell-free circulating tumor DNA (ctDNA). The aim of this study is to overcome the lack of sensitivity and specificity of current microarray approaches introducing a rapid and sensitive microarray-based assay for the multiplex detection of minority mutations of oncogenes (KRAS, NRAS and BRAF) with relevant diagnostics implications in tissue biopsies and plasma samples in metastatic colorectal cancer patients. In our approach, either wild-type or mutated PCR fragments are hybridized in solution, in a temperature gradient, with a set of reporters with a 5' domain, complementary to the target sequences and a 3' domain complementary to a surface immobilized probe. Upon specific hybridization in solution, which occurs specifically thanks to the temperature gradients, wild-type and mutated samples are captured at specific location on the surface by hybridization of the 3' reporter domain with its complementary immobilized probe sequence. The most common mutations in KRAS, NRAS and BRAF genes were detected in less than 90 minutes in tissue biopsies and plasma samples of metastatic colorectal cancer patients. Moreover, the method was able to reveal mutant alleles representing less than 0,3% of total DNA. We demonstrated detection limits superior to those provided by many current technologies in the detection of RAS and BRAF gene superfamily mutations, a level of sensitivity compatible with the analysis of cell free circulating tumor DNA in liquid biopsy.

Next-generation sequencing approach for the diagnosis of human diseases: open challenges and new opportunities.

The rapid evolution and widespread use of next generation sequencing (NGS) in clinical laboratories has allowed an incredible progress in the genetic diagnostics of several inherited disorders. However, the new technologies have brought new challenges. In this review we consider the important issue of NGS data analysis, as well as the interpretation of unknown genetic variants and the management of the incidental findings. Moreover, we focus the attention on the new professional figure of bioinformatics and the new role of medical geneticists in clinical management of patients. Furthermore, we consider some of the main clinical applications of NGS, taking into consideration that there will be a growing progress in this field in the forthcoming future.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma.

BACKGROUND: Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ. METHODS: Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing ß-thalassaemia and 7 cystic fibrosis. RESULTS: In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for ß-thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian ß-thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for ß-thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures. CONCLUSIONS: COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed.

DNA microarray-based solid-phase PCR on copoly (DMA-NAS-MAPS) silicon coated slides: An example of relevant clinical application.

In a previous study we developed a highly sensitive DNA microarray for the detection of common KRAS oncogenic mutations, which has been proven to be highly specific in assigning the correct genotype without any enrichment strategy even in the presence of minority mutated alleles. However, in this approach, the need of a spotter for the deposition of the purified PCR products on the substrates and the purification step of the conventional PCR are serious drawbacks. To overcome these limitations we have introduced the solid-phase polymerase chain reaction (SP-PCR) to form the array of PCR products starting from the oligonucleotide primers. This work was possible thanks to the great thermal stability of the copoly (DMA-NAS-MAPS) coating which withstands PCR thermal cycling temperatures. As an example of the application of this platform we performed the analysis of six common mutations in the codon 12 of KRAS gene (G12A, G12C, G12D, G12R, G12S, and G12V). In conclusion solid-phase PCR, combined with dual-color hybridization, allows mutation analysis in a shorter time span and is more suitable for automation.