3’ isomiR species and DNA contamination influence reliable quantification of microRNAs by stem-loop quantitative PCR

MicroRNAs (miRNAs) are ∼20-24 nucleotide-long regulatory RNAs that have been proven to play important roles in many cellular processes. Since their discovery, a number of different techniques have been developed to detect and accurately quantify them. For individual mature miRNA measurements, quantitative stem-loop real-time PCR represents a widely used method. Although there are some data on optimization of this technique, there are still many factors that have not been investigated yet. In this study, we have thoroughly optimized this technique and pointed out several important factors that influence reliable quantification. First, we found that total RNA input can affect the measurements. Second, our data showed that carryover DNA contamination could also mislead the detection in a sequence-specific manner. Additionally, we provided evidence that different 3' isomiR species of a particular miRNA can be reverse transcribed and cross-detected even by specifically targeted assays. Besides these, we have investigated the measurement of reaction efficiencies from total RNA samples and the accuracy of simultaneous reverse transcription reactions for increasing reliability and cost effectiveness without the loss of sensitivity and specificity. In summary, we provide a detailed, refined protocol for reliable detection of microRNA species by quantitative stem-loop PCR

One locus, several functional RNAs—emerging roles of the mechanisms responsible for the sequence variability of microRNAs

With the development of modern molecular genetics, the original “one gene-one enzyme” hypothesis has been outdated. For protein coding genes, the discovery of alternative splicing and RNA editing provided the biochemical background for the RNA repertoire of a single locus, which also serves as an important pillar for the enormous protein variability of the genomes. Non-protein coding RNA genes were also revealed to produce several RNA species with distinct functions. The loci of microRNAs (miRNAs), encoding for small endogenous regulatory RNAs, were also found to produce a population of small RNAs, rather than a single defined product. This review aims to present the mechanisms contributing to the astonishing variability of miRNAs revealed by the new sequencing technologies. One important source is the careful balance of arm selection, producing sequentially different 5p- or 3p-miRNAs from the same pre-miRNA, thereby broadening the number of regulated target RNAs and the phenotypic response. In addition, the formation of 5', 3' and polymorphic isomiRs, with variable end and internal sequences also leads to a higher number of targeted sequences, and increases the regulatory output. These miRNA maturation processes, together with other known mechanisms such as RNA editing, further increase the potential outcome of this small RNA pathway. By discussing the subtle mechanisms behind the sequence diversity of miRNAs, this review intends to reveal this engaging aspect of the inherited “RNA world”, how it contributes to the almost infinite molecular variability among living organisms, and how this variability can be exploited to treat human diseases. © 2023, The Author(s)

Transcription activity of transposon sequence limits Sleeping Beauty transposition.

Sleeping Beauty (SB) transposon based technology has been extensively applied in basic research and biotechnology for routine cell culture gene delivery and vertebrate transgenesis, and it is also investigated in various gene therapy applications. Cell tolerance for the transgene is a key factor during transgenesis and is modulated not only through the type but by the dose of expression. Our experimental results exemplify that transgenes regulated with high activity promoters can reduce the overall success of gene delivery. Observations connected to transposon donors regulated by different promoters have also revealed inverse correlation between transcription activity and the hyperactive variant SB100X excision efficiency. This competition between transcription and transposition was independent of the transgene coding sequence and did not alter the transgenic efficiency in general. However, promoters applied in the transgene cassette can produce different average copy numbers depending on the transcriptional activity of the transposon. Unlike the piggyBac (PB) transposon system, this phenomenon allows a fine balance of expression using the high copy potential SB system that adjusts the copy number of lower activity promoter driven transgenes to a higher expression level. All this contributes to a well-tolerated and satisfactory transgenesis, and would be important to consider in gene therapy applications

Excision efficiency is not strongly coupled to transgenic rate: cell type dependent transposition efficiency of Sleeping Beauty and piggyBac DNA transposons

The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, “overproduction inhibition,” a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own “in-house” platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2–3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future

A molekuláris onkogenezis mechanizmusai gyakori daganatokban = Mechanisms of molecular oncogenesis in common malignancies

Munkacsoportunk két évtizede megkezdett kutatási programját folytatta, amelynek fő célkitűzése új molekuláris rákgenetikai ismeretek szerzése a rák iránti fokozott genetikai fogékonyság molekuláris tényezőinek megismerésére. Vizsgálataink a Magyarországon gyakori daganatos megbetegedésekre (emlőrák, vastagbélrák, ivarszervi daganatok), elsősorban ezek familiáris formáira irányultak. Folytattuk, illetve megkezdtük a daganatszindrómák hajlamosító génjeinek elemzését örökletes emlő- és petefészekrákokban (BRCA1/2, CHEK2), familiáris adenomatozus polyposisban (APC), és herediter, nem a polyposis talaján kialakuló vastagbélrák szindróma (HNPCC) "mutátor gén"-jeiben (MSH2, MLH1). Az emlőrákos családokon, valamint kohorszokon nyert kutatási eredmények adatainak nemzetközi szintű összesítése, molekuláris epidemiológiai kiértékelése révén az örökletes daganatok kialakulására hajlamosító mutációt hordozók rákkockázatáról, a prevenciós tényezőkről és a betegség genetika-klinikai-pathológiai összefüggéseiről szereztünk további új ismereteket. Nemzetközi együttműködésben hozzájárultunk a csírasejtes hererákra hajlamosító első genetikai variánsok azonosításához. Daganatos megbetegedésre hajlamosító gének (BRCA1, STK11) esetében elemeztük az alternatív splicing szerepét a betegség kialakulásában. Újabban megkezdett génexpressziós profilvizsgálatok révén bepillantást nyertünk a molekuláris genetikai útvonalak és a metabolikus útvonalak kölcsönhatásába. | We have extended our molecular cancer genetic studies that were initiated in Hungary two decades ago and were aimed at providing new knowledge on molecular cancer genetics, with a focus on genetic susceptibility to cancer. The studies were conducted on common malignancies in Hungary (breast, colorectal and genitourinary cancers). Extended analysis of the predisposing genes of cancer syndromes was initiated or continued for hereditary breast- and/or ovarian cancer (BRCA1, BRCA2, CHEK2), for familial adenomatous polyposis (APC), for Peutz-Jeghers syndrome (STK11), and for hereditary non-polyposis colorectal carcinoma, HNPCC ("mutator genes" such as MSH2, MLH1). Forwarding the result and data of our molecular genetic analysis on breast cancer families and cancer cohorts for international data integration and molecular epidemiological analysis resulted in generation of new knowledge on cancer risk for the carriers of deleterious germ-line mutations, and on genetic-clinical and pathological correlations in development of breast cancer. Participating in international studies we have contributed to identification of new genetic variants predisposing to germ cell testicular cancer. The role of alternative splicing in development of cancer was investigated in cancer susceptibility genes BRCA1 and STK11. By recent introduction of gene-expression profiling we have gained preliminary insight into the interaction of metabolic and molecular pathways

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications

There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the DeltaDeltaCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies

Dissociative recombination of N2_2H+^+: A revisited study

Dissociative recombination of N$_2$H$^+$ is explored in a two-step theoretical study. In a first step, a diatomic (1D) rough model with frozen NN bond and frozen angles is adopted, in the framework of the multichannel quantum defect theory (MQDT). The importance of the indirect mechanism and of the bending mode is revealed, in spite of the disagreement between our cross section and the experimental one. In a second step, we use our recently elaborated 3D approach based on the normal mode approximation combined with R-matrix theory and MQDT. This approach results in satisfactory agreement with storage-ring measurements, significantly better at very low energy than the former calculations.Comment: 9 pages, 5 figures, 1 tabl