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BDNF Val66Met Polymorphism, the Allele-Specific Analysis by QRT-PCR – a Novel Protocol

Molecular Blood Group Screening in Donors from Arabian Countries and Iran Using High-Throughput MALDI-TOF Mass Spectrometry and PCR-SSP

Background and aims: Only little is known about blood groups other than ABO blood groups and Rhesus factors in Arabian countries and Iran. During the last years, increased migration to Central Europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. Therefore, blood group allele frequencies should be determined in individuals from Arabian countries and Iran by molecular typing and compared to a German rare donor panel.
Methods: 1,111 samples including 800 individuals from Syria, 147 from Iran, 123 from the Arabian Peninsula, and 41 from Northern African countries were included in a MALDI-TOF MS assay to detect polymorphisms coding for Kk, Fy(a/b), Fy<sub>null</sub>, C<sub>w</sub>, Jk(a/b), Jo(a+/a-), Lu(a/b), Lu(8/14), Ss, Do(a/b), Co(a/b), In(a/b), Js(a/b), Kp(a/b), and variant alleles RHCE*c.697C>G and RHCE *c.733C>G. Yt(a/b), S-s-U-, Vel<sub>null</sub>, Co<sub>null</sub>, and RHCE *c.667G>T were tested by PCR-SSP.
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Results: </strong> Of the Arabian donors, 2% were homozygous for the <i>FY</i> *<i>02.01N</i> allele (Fy<sub>null</sub>), and 15.7% carried the heterozygous mutation. However, 0.8% of the German donors also carried 1 copy of the allele. 3.6% of all and 29.3% of Northern African donors were heterozygous for the <i>RHCE</i> *<i>c.733C>G</i> substitution, 0.4% of the Syrian probands were heterozygous for <i>DO</i> *<i>01/DO</i> *<i>01.-05</i>, a genotype that was lacking in German donors.

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Whereas the <i>KEL</i> *<i>02.06</i> allele coding for the Js(a) phenotype was missing in Germans; 0.8% of the Syrian donors carried 1 copy of this allele. 1.8% of the Syrian but only 0.3% of the German donors were negative for <i>YT</i> *<i>01</i>. One donor from Northern Africa homo-zygously carried the <i>GYPB</i> *<i>270+5g>t</i> mutation, inducing the S-s-U+<sup>w</sup> phenotype, and in 2 German donors a <i>GYPB</i> *<i>c.161G>A</i> exchange, which induces the Mit+ phenotype, caused a <i>GYPB</i> *<i>03</i> allele dropout in the MALDI assay. The overall failure rate of the Arabian panel was 0.4%.

CRISPR-mediated knock-in in the mouse embryo using long single stranded DNA donors synthesised by biotinylated PCR

Successful gene knock-in by CRISPR-Cas9 in the mouse zygote requires three components; guideRNA, Cas9 protein and a suitable donor template, which usually comprises homology flanked insert sequence. Recently, long single stranded DNA (lssDNA) donors have emerged as a popular choice of DNA donor, outperforming dsDNA templates in terms of knock-in efficiency for gene tagging and generating conditional alleles.

 

The generation of these donors can be achieved through several methods that may introduce errors in the sequence, result in poor yields, and contain dsDNA contamination. We have developed our own cost-effective lssDNA synthesis methodology that results in high purity, sequence verified, low contamination lssDNA donors. We provide a detailed methodology on the design and generation of such donors for gene tagging experiments and generating conditional alleles.

BDNF Val66Met Polymorphism, the Allele-Specific Analysis by qRT-PCR – a Novel Protocol

Background: Alteration in brain-derived neurotrophic factor (BDNF) production is a marker of neuropathological conditions, which has led to the investigation of Val66Met polymorphism occurring in the human BDNF gene (BDNF). Presently, there are no reported methods available for the analysis of Val66Met impact on human BDNF functioning.

Purpose: To develop a qRT-PCR protocol for the allele-specific expression evaluation of the Val66Met polymorphism in BDNF

Methods: Using RNA extracted from muscle samples of 9 healthy volunteers (32.9 ± 10.3 y) at rest and following a maximal effort aerobic capacity exercise test, a protocol was developed for the detection of Val66/Met66 allele-specific BDNF expression in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) – relative to housekeeping genes – and validated by absolute quantification in Droplet Digital Polymerase Chain Reaction (ddPCR).

 

Results: Differences in the relative values of BDNF mRNA were confirmed by ddPCR analysis. HPRT1 and B2M were the most stable genes expressed in muscle tissue among different metabolic conditions, while GAPDH revealed to be metabolic responsive. Conclusion: Our qRT-PCR protocol successfully determines the allele-specific detection and changes in BDNF expression regarding the Val66Met polymorphism.

Standardization of BCR-ABL1 p210 Monitoring: From Nested to Digital PCR

The introduction of tyrosine kinase inhibitors in 2001 as a targeted anticancer therapy has significantly improved the quality of life and survival of patients with chronic myeloid leukemia. At the same time, with the introduction of tyrosine kinase inhibitors, the need for precise monitoring of the molecular response to therapy has emerged. Starting with a qualitative polymerase chain reaction, followed by the introduction of a quantitative polymerase chain reaction to determine the exact quantity of the transcript of interest-p210 BCR-ABL1, molecular monitoring in patients with chronic myeloid leukemia was internationally standardized.

 

This enabled precise monitoring of the therapeutic response, unification of therapeutic protocols, and comparison of results between different laboratories. This review aims to summarize the steps in the diagnosis and molecular monitoring of p210 BCR-ABL1, as well as to consider the possible future application of a more sophisticated method such as digital polymerase chain reaction.

Droplet Digital PCR Analysis of Liquid Biopsy Samples Unveils the Diagnostic Role of hsa-miR-133a-3p and hsa-miR-375-3p in Oral Cancer

Despite the availability of screening programs, oral cancer deaths are increasing due to the lack of diagnostic biomarkers leading to late diagnosis and a poor prognosis. Therefore, there is an urgent need to discover novel effective biomarkers for this tumor. On these bases, the aim of this study was to validate the diagnostic potential of microRNAs (miRNAs) through the analysis of liquid biopsy samples obtained from ten oral cancer patients and ten healthy controls. The expression of four selected miRNAs was evaluated by using droplet digital PCR (ddPCR) in a pilot cohort of ten oral cancer patients and ten healthy donors. Bioinformatics analyses were performed to assess the functional role of these miRNAs.

 

The expression levels of the predicted down-regulated hsa-miR-133a-3p and hsa-miR-375-3p were significantly reduced in oral cancer patients compared to normal individuals while no significant results were obtained for the up-regulated hsa-miR-503-5p and hsa-miR-196a-5p. ROC analysis confirmed the high sensitivity and specificity of hsa-miR-375-3p and hsa-miR-133a-3p. Therefore, both miRNAs are significantly down-regulated in cancer patients and can be used as biomarkers for the early diagnosis of oral cancer. The analysis of circulating miRNAs in a larger series of patients is mandatory to confirm the results obtained in this pilot study.

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