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Multiplex Testing of Bcr-Abl1 and Jak2 V617f in Suspected Mpn Using Rt-Pcr Rdb Method
Published in Cut Adeya Adella, Stem Cell Oncology, 2018
N. Masykura, F. Albertha, A.R.H. Utomo, U. Habibah, M. Yunus, Suharsono, F. Selasih, A. Bowolaksono
RT reverse transcriptase cDNA conversion. Isolated RNAs were converted into cDNA with reverse transcriptase enzyme and random hexamer primers using Transcriptor First Strand cDNA Synthesis (Roche).
Toxicogenomic and Toxicoproteomic Approaches for Biomarkers
Published in Anthony P. DeCaprio, Toxicologic Biomarkers, 2006
The powerful PCR technique rapidly became incorporated into other genomic approaches in addition to its direct use for analyzing expression of individual genes. One of these applications was in a method called differential display of mRNA expression (33). This approach to genomic screening exploited a variation of multiplex PCR and was used both to globally examine patterns of altered gene expression and for direct gene discovery. The technique is based on use of short random hexamer primers that simultaneously amplify numerous segments of complementary DNA (cDNA) in the entire isolated RNA pool. Parallel amplification of RNAs from control and treated samples is followed by running out the PCR products on a sequencing gel for a side-by-side pattern comparison of the two samples. Typically, it was observed that most bands were identical in the two samples, indicating that most mRNAs were not differentially expressed (Fig. 4). This observation also provides a means for normalizing the treated to the control pattern, such that the altered band intensities are more apparent, and represent only those products that are substantially up or downregulated by a given treatment. The mRNA segment of interest can then be identified by subsequent isolation of the band from the gel, followed by reamplification, subcloning, and sequencing of the product. Figure 4 shows an example of the results of a differential display experiment from our laboratory, examining the effects of cadmium on gene expression in the zooplankton, Daphnia pulex. Using this method, we identified several candidate mRNA fragments that were up- or downregulated in response to nonovertly toxic exposures of Daphnia to cadmium. As expected, one of these fragments turned out to represent the mRNA for Daphnia metallothionein, but several other cadmium-responsive mRNAs were also identified.
Methods in Molecular Biology
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
A large percentage of DNA sequence in eukaryotes is not expressed. Thus, expressed sequences can be identified more easily by working with complimentary DNA (cDNA). Additionally, cDNA sequences are smaller in size and can be cloned more efficiently into a variety of vectors. The first step of cDNA synthesis depends on an enzyme called RNA-dependent DNA polymerase or reverse transcriptase (Fig. 3). Reverse transcription exploits a characteristic of mature mRNAs known as the 3′ -polyadenylated region, commonly called the poly (A) tail, as a common binding site for poly (T) DNA primers. These primers will anneal to the 3′ end of every mRNA in the solution, allowing 5′ to 3′ synthesis of cDNA by the reverse transcriptase enzyme. Gene-specific primers or random hexamer primers can also be used to generate the cDNA. The mRNA is subsequently removed by treatment with RNase H. The second strand of the cDNA is usually synthesized by a combined action of both DNA polymerase 1 and T4 DNA polymerase, which used the RNA fragments as amorces. DNA polymerase I is used since the 5′ → 3′ exonuclease activity is needed to remove RNA in front of the enzyme. DNA polymerase I also removes the RNA primer from the 5′ lagging strand and fills in the recessed 3′ end. T4 DNA ligase then joins the various fragments together into a continuous strand of DNA. The final products are cDNA molecules with blunt ends, which can be further ligated to linkers containing restriction sites using T4 DNA ligase. The cDNA molecules are ultimately cleaved at the restriction site of the linker and ligated into a compatible vector for the formation of a cDNA library. The ligation mixture is transformed into a bacterial or phage host. A cDNA library can be used for different purposes. There is a broad diversity of cDNA libraries that are available commercially. As cDNA sequences differ in each library, comparison between libraries constructed from cells derived from different organisms can provide useful information.
Overview of gene expression techniques with an emphasis on vitamin D related studies
Published in Current Medical Research and Opinion, 2023
Jeffrey Justin Margret, Sushil K. Jain
Recent technologies have made it possible to achieve a detailed understanding of the genomic architecture and complex nature of gene expression patterns across various cell types. However, these technological advances also raise several concerns regarding the mechanistic aspects of gene regulation and genome function. They continue to generate vast amounts of complex data pertaining to gene expression and regulation in a range of biological contexts42. Retrieving these data from the database, interpreting them, and then storing them continues to be the main challenge. Variations in GC contents, random hexamer primer binding, and differences in transcript length add to the limitations26. Measurement of genome-wide data suffer from technical limitations including noise and biases. Technical or biological replicates do not yield similar readings due to noise, which affects the precision of the data across or within gene correlations. An effective estimate can be made by analyzing multiple technical and biological replicates, but bias affects the accuracy of the data being over or underestimated3.
Oncolytic adenovirus promotes vascular normalization and nonclassical tertiary lymphoid structure formation through STING-mediated DC activation
Published in OncoImmunology, 2022
Teng He, Zhixing Hao, Mingjie Lin, Zhongwei Xin, Yongyuan Chen, Wei Ouyang, Qi Yang, Xiaoke Chen, Hui Zhou, Wanying Zhang, Pin Wu, Feng Xu
Total RNA was extracted from frozen treated cells by the TRIzol-chloroform extraction method. Subsequently, the isolated total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). mRNA was purified by Oligo(dT)-attached magnetic beads and fragmented at RT. First-strand cDNA was generated by random hexamer-reverse transcription, and second-strand cDNA was synthesized. Then, the generated cDNA was amplified and purified with Ampure XP Beads, and the final library was obtained by splint oligonucleotide sequence circulation. In vitro, sscir cDNA was rolled and amplified under the action of phi29. When it was amplified to 100–1000 copies, it formed a DNA nanoball (DNB) and was run on a Bgiseq500 sequencing platform. Bgiseq500 contains patterned nanoarrays, which are a kind of binding site array chip formed and processed on the surface of a silicon wafer. The spacing of modification sites on the chip is uniform, and only one DNB is fixed at each site, to ensure that the optical signals of each DNB will not interfere with those of the others and to improve the accuracy of subsequent sequencing. Subsequently, a single-ended 50-base reading was generated on the Bgiseq500 platform (BGI, Shenzhen, China).
Inhibition of Circ-Snrk ameliorates apoptosis and inflammation in acute kidney injury by regulating the MAPK pathway
Published in Renal Failure, 2022
Fanhang Meng, Qiuyuan Chen, Shijie Gu, Ruiwen Cui, Qing Ma, Ronghua Cao, Ming Zhao
The NRK-52E cells underwent transfection with siRNA-1 or siRNA-NC and then were treated with H/R. RNA was isolated and enriched by magnetic bead Oligo-dT (Dynal Biotech, Oslo, Norway). After RNA fragmentation, [the RNA samples underwent] random hexamer primed reverse transcription and adapter ligation. The generated libraries were examined by Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Germany) and subjected to sequencing on an Illumina HiSeq2000 instrument (Illumina, San Diego, CA). Next, the clean data was obtained after raw data filtering, and aligned to the hg19 human reference genome using Short Oligonucleotide Analysis Package alignment software. Read counts were analyzed, and differentially expressed mRNAs were screened based on P-value (<0.05) and absolute value of fold change (|FC|) >1.5. Hierarchical cluster analysis was created by the heatmap package in R software and the scatter plot was drawn using R software.