The authors would like to acknowledge prof. J.J. McGuire, prof. R. Kofler and dr. K. Quint for providing the resistant cell lines used in this work. The study has been founded by grants from Cancer Center Amsterdam (CCA) Foundation (to JC, EG and RS), the KiKa (Children Cancer-free grant for AW) foundation, the Law Offices of Peter G. Angelos Grant from the Mesothelioma Applied Research Foundation (to VEG and EG), Associazione Italiana per la Ricerca sul Cancro (AIRC), Istituto Toscano Tumori (ITT), and Regione Toscana Bando FAS Salute (to EG).

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The authors have nothing to disclose.

Here we describe a novel approach that combines well-established cytotoxicity screening techniques and powerful NGS-based transcriptomic analyses to identify differential splicing events in relation to drug resistance. Spectrophotometric assays are convenient and robust high-throughput methods to assess drug sensitivity in in vitro cancer models and represent the first choice for many laboratories performing cytotoxicity screenings. Troubleshooting as well as possible variations for this method were extensively ...

Despite considerable advances in cancer treatment, resistance of malignant cells to chemotherapy, either intrinsic or acquired upon prolonged drug exposure, is the major reason for treatment failure in a wide range of leukemia and solid tumors1.
In order to delineate the mechanisms underlying drug resistance, in vitro cell line models are developed by stepwise selection of cancer cells resistant to chemotherapeutic agents. This procedure mimics the regimes used in the clinical settings and therefore allows in depth investigation of relevant resistance mechanisms. Resistant cells which survive the treatment are then distinguished from parental sensitive cells by using cell viability/cytotoxicity assays2. In vitro drug resistance profiles of primary cells have been shown to be significantly related to clinical response to chemotherapy3.
High-throughput cytotoxicity assays constitute a convenient method to determine drug sensitivity in vitro. Herein, the viability of cells is assessed by for instance the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide - MTT assay4, which is based on metabolic conversion of certain substrates (i.e., tetrazolium salts) into colored products, thereby reflecting the mitochondrial activity of cells. Alternatively, the cellular protein content can be quantified using the sulforhodamine B (SRB) assay5. Here, the number of viable cells is proportional to the optical density (OD) measured at an appropriate wavelength in a spectrophotometer, with no need of extensive and time-consuming cell counting procedures. The growth inhibition induced by a certain chemotherapeutic drug can be calculated based on the OD of the wells in which cells were treated with a test agent and compared with the OD of untreated control cells. A dose-response curve is obtained by plotting drug concentrations versus percentages of viable cells relative to control cells. Finally, drug sensitivity can be reported as the concentration that results in 50% of cell growth inhibition as compared to untreated cells (IC50).
The mechanisms underlying drug resistance include many different abnormalities, such as alterations affecting gene expression of determinants of drug activity and cellular metabolism. These molecular lesions, including mutations, aberrations at a transcriptional and post-transcriptional level as well as disturbed epigenetic regulation often affect genes involved either in drug metabolism or apoptosis6.
Alternative pre-mRNA splicing and its intricate regulation have recently received considerable attention as a novel entity that may dictate drug resistance of cancer cells7. Up to 95% of human genes are alternatively spliced in normal cells by means of this tightly regulated process which produces many different protein isoforms from the same gene. Alternative splicing is often deregulated in cancer and several tumors are characterized by altered splicing of a growing number of genes involved in drug metabolism (i.e., deoxycytidine kinase, folylpolyglutamate synthetase, or Multidrug resistance proteins)6,8. However, comprehensive analysis of splicing profiles of drug resistant cells is painfully lacking. Therefore, it is imperative to develop high throughput methods for alternative splicing analysis. This could help to develop more effective therapeutic approaches.
During the last decade, the rapid development of next generation sequencing (NGS) technologies has enriched biomedical research with new insights into the molecular mechanisms governing regulation of genome expression and their role in various biological processes9. RNA-sequencing (RNA-seq) is a powerful sub-application of NGS in the field of transcriptomics. It allows a genome-wide profiling (both qualitatively and quantitatively) of the expression patterns of thousands of genes simultaneously and is well suited for the characterization of novel coding mRNAs as well as long non-coding RNA, miRNA, siRNA, and other small RNA classes (e.g., snRNA and piRNA)10,11.
RNA-Seq has many advantages over previous technologies for transcriptome characterization (e.g., Sanger sequencing and expression microarrays). It is not based on existing genome annotation, it has a single-nucleotide level of resolution and it has a broader dynamic range for expression level estimation. Briefly, the basic experimental workflow of RNA-seq experiments consists of polyadenylated transcript (mRNA) selection and fragmentation, followed by conversion into cDNA, library construction and finally, massively parallel deep sequencing12,13. Due to rapid drop of sequencing costs over the last few years, RNA-seq is gradually replacing other technologies and significant efforts are being made to improve the library preparation protocols. For instance, it is now possible to retain the strand information of mRNA transcripts by marking the second strand cDNA with deoxyuridine triphosphate (dUTP) and, prior to PCR amplification, digesting the marked strand with uracil-DNA-glycosilase (UDG). This process enhances the accuracy of gene annotation and estimation of the expression levels14,15.
The analysis and interpretation of RNA-seq data require complex and powerful computational software packages and processing within bioinformatic pipelines16,17. First, the raw reads undergo quality control by removing technical and biological artefacts and discarding (trimming) the sequences which do not reach stringent quality requirements. Subsequently the reads for each sample are mapped to a reference genome and indexed into gene-level, exon-level, or transcript-level, in order to determine the abundance of each category. Depending on the application, refined data are then computed through statistical models for the identification of allele-specific expression, alternative splicing, gene fusions and single nucleotide polymorphisms (SNPs)12. Finally, differential analysis on selected level (i.e., gene expression or alternative splicing) can be used to compare samples obtained under different conditions.
Differential splicing analysis describes the differences in splice site usage between two samples. An increasing number of software packages devoted to this purpose are available based on different statistical models, performances and user interface18. Among these, MATS (Multivariate Analysis of Transcript Splicing) emerges as a freely available and precise computational tool based on a Bayesian statistical framework and designed to detect differential splicing events from either single or paired end RNA-seq data. Starting from the aligned (.bam) files, MATS can detect all major types of alternative splicing events (exon skipping, alternative 3&#39; splice site, alternative 5&#39; splice site, mutually exclusive exons and intron retention - also see Figure 1).
First, the software identifies reads which support a certain splice event, for instance exon skipping, and classifies them into two types. &#34;Inclusion reads&#34; (for the canonical splice event) map within the investigated exon and span the junctions between that specific exon and the two upstream and downstream flanking exons. &#34;Skipping reads&#34; (for the alternative splice event) span the junction between the two flanking exons. Subsequently, MATS returns the normalized inclusion level for both the canonical and alternative events and compares values between samples or conditions. Ultimately, it calculates P-value and false discovery rate (FDR) assuming that the difference in the variant ratio of a gene between two conditions exceeds a given user-defined threshold for each splicing event19,34.
Following differential splicing analysis in conjunction with RNA-seq, an extensive experimental validation is warranted in order to identify true-positive gene candidates18. Quantitative reverse transcribed-polymerase chain reaction (qRT-PCR) is the most commonly used and optimal method in validation of candidates obtained from RNA-Seq analysis20. The aim of this paper is to provide a robust methodology to investigate drug resistance-related splicing profiles in solid tumors and hematological malignancies. Our approach utilizes RNA-seq-based transcriptome profiling of selected cell line models of drug resistant cancers in combination with an established qRT-PCR method for the validation of candidate genes implicated in drug resistance.
The human leukemia cell line models used in this study included pediatric T-cell acute lymphoblastic leukemia (T-ALL) cell line CCRF-CEM (CEM-WT), its two glucocorticoid (GC)-resistant subclones CEM-C7H2-R5C3 (CEM-C3) and CEM-C7R5 (CEM-R5)21,22 and the methotrexate (MTX)-resistant subline CEM/R30dm23. Although current therapies based on GCs and MTX establish clinical benefit in about 90% of cases, the emergence of GC-resistance still represents an unsolved problem with an unclear molecular mechanism. To isolate GC-resistant sub-clones, CEM-WT cells were cultured in 1 &#181;M dexamethasone (Dex) for 2 to 3 weeks. MTX-resistant subline CEM/R30dm was developed through repeated short- term (24 hr) exposure of CEM-WT cells to 30 &#181;M MTX as a mimic of clinical protocols. Interestingly, this cell line also displayed cross-resistance to Dex (unpublished results) for which the mechanism is not fully understood.
The solid tumor model investigated in the present study is pancreatic ductal adenocarcinoma, notorious for its extraordinary refractoriness to chemotherapy. To this end, we selected Panc-1 cell line and its gemcitabine-resistant sub-clone Panc-1R obtained by continuous incubation with 1 &#181;M of the drug24. Here we describe an approach to discover novel mechanisms underlying in-vitro drug resistance by combining three protocols: colorimetric cytotoxicity assays to assess drug sensitivity in leukemic cells and cancer cells from solid tumors, RNA-seq-based pipeline to identify novel splice variants related to drug sensitivity/resistance and RT-PCR and qRT-PCR analysis to validate potential candidates.

<table><tbody><tr><td>Sulforhodamine B</td><td>Sigma-Aldrich</td><td>230162</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Sigma-Aldrich</td><td>251399</td><td></td></tr><tr><td>CCRF-CEM</td><td>ATCC, Manassas, VA, USA</td><td>ATCC CCL-119</td><td></td></tr><tr><td>Panc-1</td><td>ATCC, Manassas, VA, USA</td><td>ATCC CRL-1469</td><td></td></tr><tr><td>DMEM high glucose</td><td>Lonza, Basel, Switzerland</td><td>12-604F</td><td></td></tr><tr><td>RPMI-1640&amp;nbsp;</td><td>Gibco, Carlsbad, CA, USA</td><td>11875093</td><td></td></tr><tr><td>Fetal bovine and calf serum</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>758093</td><td></td></tr><tr><td>penicillin G streptomycin sulphate</td><td>Gibco, Carlsbad, CA, USA</td><td>15140122</td><td></td></tr><tr><td>Tris(hydroxymethyl)-aminomethane</td><td>Sigma Aldrich</td><td>252859</td><td></td></tr><tr><td>MTT formazan</td><td>Sigma Aldrich</td><td>M2003</td><td></td></tr><tr><td>Anthos-Elisa-reader 2001</td><td>Labtec, Heerhugowaard, Netherlands</td><td></td><td>UV-Vis 96-well plate spectrophotometer</td></tr><tr><td>Greiner CELLSTAR 96 well plates</td><td>Greiner/Sigma</td><td>M0812-100EA</td><td></td></tr><tr><td>Trypsin/EDTA Solution 100 ml</td><td>Lonza, Basel, Switzerland</td><td>CC-5012</td><td></td></tr><tr><td>CELLSTAR Cell Culture Flasks 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>82051-074</td><td></td></tr><tr><td>CELLSTAR Cell Culture Flasks 75 cm&lt;sup&gt;3&lt;/sup&gt;</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>82050-856</td><td></td></tr><tr><td>Phosphate Buffered Saline (NaCl 0.9%)</td><td>B.Braun Melsungen AG, Germany</td><td>362 3140</td><td></td></tr></tbody></table>

using rna-sequencing to detect novel splice variants related to drug resistance in in vitro cancer models

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.

The cytotoxicity assays described in the protocol provide a reliable and robust method to assess the resistance of cancer cells to chemotherapeutic agents in vitro. By means of the MTT assay, sensitivity to Dex was determined in four T-ALL cell lines, including Dex-sensitive parental CEM-WT cells, and three Dex-resistant sublines: CEM/R30dm, CEM-R5 and CEM-C3. Two different concentration ranges had to be used due to the large difference in sensitivity between CEM-WT (2 &#181;M - ...

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

Here we describe a protocol 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 parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

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

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

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Research

JoVE Journal

Cancer Research

13.6K Views.  VU University Medical Center.  Hier beschrijven we een protocol gericht op onderzoek naar de gevolgen van afwijkende splicing op resistentie in solide tumoren en hematologische maligniteiten. Om dit doel te bereiken, analyseerden we het transcriptoom profielen van de ouders en bestand in vitro modellen door middel van RNA-seq en vestigde een qRT-PCR-methode om kandidaatgenen te valideren. 

Video: Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Genetics

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

Optimizing Low-Frequency Mutation Detection with Wild-Type Blocking PCR

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Biology

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Biochemistry

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Medicine

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

SNP-Guided 5-FU Therapy in Gastric Cancer Using Ion Torrent Sequencing

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although 5-fluorouracil (5-FU) is widely used for treatment, its therapeutic sensitivity and drug tolerance still need to be determined, which emphasizes the importance of individualized administration. Pharmacogenetics can guide the clinical implementation of individualized treatment. Single nucleotide polymorphisms (SNPs), as a genetic marker, contribute to the selection of appropriate chemotherapy regimens and dosages. Some SNPs are associated with folate metabolism, the therapeutic target of 5-FU. Methylenetetrahydrofolate reductase (MTHFR) rs1801131 and rs1801133, dihydrofolate reductase (DHFR) rs1650697 and rs442767, methionine synthase (MTR) rs1805087, gamma-glutamyl hydrolase (GGH) rs11545078 and solute carrier family 19 member 1 (SLC19A1) rs1051298 have been investigated in different kinds of cancers and antifolate antitumor drugs, which have potential forecasting and guiding significance for application of 5-FU. The ion torrent next-generation semiconductor sequencing technology can rapidly detect gastric cancer-related SNPs. Each time a base is extended in a DNA chain, an H+ will be released, causing local pH changes. The ionic sensor detects pH changes and converts chemical signals into digital signals, achieving sequencing by synthesis. This technique has low sample requirement, simple operation, low cost, and fast sequencing speed, which is beneficial for guiding individualized chemotherapy by SNPs.

Author Spotlight: Genetic Profiling for Fluorouracil Response in Gastric Cancer

This protocol proposes holistic laboratory procedures required to detect single nucleotide polymorphisms in gastric cancer samples based on an ion semiconductor sequencing platform. The target sequences, ligating adapters, library amplification and purification, and quality control criteria are also described in detail.

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although ...

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

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