Telomeres and Telomerase Activity in the Human Placenta

Placenta is a transient organ ensuring the intrauterine development of the individual. To meet fetal requirements, rapid and continuous cell proliferation enlarges the areas of tissues maintaining maternofetal transport. The cell division in placenta is accompanied with shortening of telomeres leading to cell senescence. Telomerase activity, on the other hand, ensures replication of telomeres and allows the organ to serve till the end of pregnancy. This balanced process may be negatively influenced by unfavorable circumstances. Here, we summarize available data on telomere length as well as telomerase activity in placentas from normal and complicated pregnancies; attention is also paid to the comparison of methods used in relevant studies

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals

PCR past, present and future

PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system

Temperature non-uniformity detection on dPCR chips and temperature sensor calibration

A microfluidic-based digital polymerase chain reaction (dPCR) chip requires precise temperature control as well as uniform temperature distribution to ensure PCR efficiency. However, measuring local temperature and its distribution over thousands of L/nL-volume samples with minimum disturbance is challenging. Here, we present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip. We filled the dPCR chip with a PCR solution containing amplified DNA fragments with a known melting temperature (TM). We then captured fluorescent images of the chip when it was heated from 70 to 99 °C, plotted the fluorescence intensity of each partition as a function of temperature, and calculated measured TM values from each partition. Finally, we created a 3-D map of the dPCR chip with the measured TM as the parameter. Even when the actual TM of the PCR solution was constant, the measured TM value varied between locations due to temperature non-uniformity in the dPCR chip. The method described here thereby characterized the distribution of temperature non-uniformity using a PCR solution with known TM as a temperature sensor. Among the non-contact temperature measurement methods, the proposed TM-based method can determine the temperature distribution within the chip, instead of only at the chip surface. The method also does not suffer from the undesirable photobleaching effect of fluorescein-based temperature measurement method. Temperature determination over the dPCR chip based on TM allowed us to calibrate the temperature sensor and improve the dPCR configuration and precision. This method is also suitable for determining the temperature uniformity of other microarray systems where there is no physical access to the system and thus direct temperature measurement is not possible

Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR

Abstract Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21

Correlation between measurements of cfDNA concentrations (<i>RHD</i> exon 5) by both methods; r = 0.9776, p = 0.000001.

<p>Correlation between measurements of cfDNA concentrations (<i>RHD</i> exon 5) by both methods; r = 0.9776, p = 0.000001.</p

Correlation between gestational age and fetal cfDNA content in maternal circulation measured by ddPCR using <i>RHD</i> exon 10<i>/GAPDH</i> ratio; r = 0.45936, p = 0.0208.

<p>Correlation between gestational age and fetal cfDNA content in maternal circulation measured by ddPCR using <i>RHD</i> exon 10<i>/GAPDH</i> ratio; r = 0.45936, p = 0.0208.</p

Performance of Droplet Digital PCR in Non-Invasive Fetal <i>RHD</i> Genotyping - Comparison with a Routine Real-Time PCR Based Approach

<div><p>Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal <i>RHD</i> genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods’ performance parameters—standard curve linearity, detection limit and measurement precision—were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438).</p></div

Statistically significant differences between the both techniques coefficients of variation (CV) for standard curve of <i>RHD</i> exon 10 replicates.

<p>A trend of increasing CVs with decreasing DNA concentrations can be seen in both methods measurement.</p