Assessment of mRNA Splice Variants by qRT-PCR

Vargas, Ileabett M. Echevarría; Vivas‐Mejía, Pablo E.

doi:10.1007/978-1-62703-547-7_13

Cited by 5 publications

(5 citation statements)

References 16 publications

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“…For exosome co-cultures, BMSCs were added to a 25-cm 2 flask, followed by addition of exosomes at 20 µg/mL. After 14 days, the expression of myocyte-specific markers (e.g., α-actin, cTnI, Cx43, and desmin) and GAPDH was measured by immunofluorescence analysis and quantitative PCR (qPCR) 16 . Briefly, total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes

Han

et al. 2018

Sci Rep

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This study aimed to investigate whether exosomes secreted by mouse GATA-4-expressing bone marrow mesenchymal stem cells (BMSCs) could induce BMSC differentiation into myocyte precursors, decrease cardiomyocyte apoptosis, and improve cardiac function following myocardial infarction (MI). BMSCs were transduced with a lentivirus carrying a doxycycline (DOX)-inducible GATA-4 or control lentivirus, and secreted exosomes from these BMSCs were collected and co-cultured with BMSCs or cardiomyocytes under hypoxic and serum free conditions. Furthermore, exosomes were injected into mice 48 h after MI. Cardiac function was evaluated by echocardiography at 48, 72, and 96 h after exosome treatment. Quantitative PCR showed that co-culture of BMSCs with GATA-4-BMSC exosomes increased cardiomyocyte-related marker expression. Co-culture of GATA-4-BMSC exosomes with cardiomyocytes in anoxic conditions decreased apoptosis as detected by flow cytometry. Injection of GATA-4-BMSC exosomes in mice 48 h after MI increased cardiac function over the next 96 h; increased cardiac blood vessel density and number of c-kit-positive cells and decreased apoptotic cardiomyocyte cells were also observed. Differential expression of candidate differentiation- and apoptosis-related miRNAs and proteins that may mediate these effects was also identified. Exosomes isolated from GATA-4-expressing BMSCs induce differentiation of BMSCs into cardiomyocyte-like cells, decrease anoxia-induced cardiomyocyte apoptosis, and improve myocardial function after infarction.

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Section: Methodsmentioning

confidence: 99%

GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes

Han

et al. 2018

Sci Rep

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“…Data was collected and analyzed using a StepOne Software v2.1 from Applied Biosystems following manufacturer’s protocol (95 °C for 2 min followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C). β-actin was used as the internal standard for the gene expression values [ 59 , 60 ]. We used the multalin software ( ) [ 61 ] for alignment analysis of miR-143-3p (mature strand) and the SLC30A8(A) fragment mRNA.…”

Section: Methodsmentioning

confidence: 99%

Targeting MicroRNA-143 Leads to Inhibition of Glioblastoma Tumor Progression

Lozada-Delgado

Grafals‐Ruiz

Miranda-Román

et al. 2018

Cancers

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Glioblastoma (GBM) is the most common and aggressive of all brain tumors, with a median survival of only 14 months after initial diagnosis. Novel therapeutic approaches are an unmet need for GBM treatment. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Several dysregulated miRNAs have been identified in all cancer types including GBM. In this study, we aimed to uncover the role of miR-143 in GBM cell lines, patient samples, and mouse models. Quantitative real-time RT-PCR of RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals. Transient transfection of GBM cells with a miR-143 oligonucleotide inhibitor (miR-143-inh) resulted in reduced cell proliferation, increased apoptosis, and cell cycle arrest. SLC30A8, a glucose metabolism-related protein, was identified as a direct target of miR-143 in GBM cells. Moreover, multiple injections of GBM tumor-bearing mice with a miR-143-inh-liposomal formulation significantly reduced tumor growth compared to control mice. The reduced in vitro cell growth and in vivo tumor growth following miRNA-143 inhibition suggests that miR-143 is a potential therapeutic target for GBM therapy.

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“…Following differential splicing analysis in conjunction with RNA-seq, an extensive experimental validation is warranted in order to identify truepositive gene candidates 18 . Quantitative reverse transcribed-polymerase chain reaction (qRT-PCR) is the most commonly used and optimal method in validation of candidates obtained from RNA-Seq analysis 20 . The aim of this paper is to provide a robust methodology to investigate drug resistance-related splicing profiles in solid tumors and hematological malignancies.…”

mentioning

confidence: 99%

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in <em>In Vitro</em> Cancer Models

Sciarrillo

Wojtuszkiewicz

Kooi

et al. 2016

JoVE

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Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

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Assessment of mRNA Splice Variants by qRT-PCR

Cited by 5 publications

References 16 publications

GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes

GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes

Targeting MicroRNA-143 Leads to Inhibition of Glioblastoma Tumor Progression

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in <em>In Vitro</em> Cancer Models

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