<p>Contributors<br>Acknowledgments<br><br>1. Past, current, and future of transcriptomics<br>Xinmin Li, Ilya Belalov, and Anton Buzdin<br><br>1.1 Introduction<br>1.2 Historical development<br>1.3 Methodological advancements<br>1.4 Microarrays<br>1.5 RNA sequencing (RNA-seq)<br>1.6 Single-cell RNA sequencing (scRNA-seq)<br>1.7 Spatial transcriptomics<br>1.8 Noncoding RNAs in gene regulation<br>1.9 MicroRNAs (miRNAs)<br>1.10 Long noncoding RNAs (lncRNAs)<br>1.11 Regulatory networks<br>1.12 Single-cell and spatial transcriptomics in understanding cellular heterogeneity<br>1.13 Single-cell transcriptomics<br>1.14 Spatial transcriptomics<br>1.15 Insights and applications<br>1.16 Challenges and future directions<br>1.17 Emerging trends and future directions in transcriptomics<br>1.18 RNA sequencing (RNA-seq) and beyond<br>1.19 Dual RNA-seq for host-pathogen interactions<br>1.20 Transcriptomics in food microbiology and agriculture<br>1.21 Multi-omics integration<br>1.22 Single-cell transcriptomics<br>1.23 Challenges and future prospects<br>1.24 Current challenges and controversies in transcriptomics<br>1.25 Technical limitations and data complexity<br>1.26 Interpretation of dual RNA-seq data<br>1.27 Environmental influences on transcriptomic profiles<br>1.28 Transcriptomics in food microbiology and safety<br>1.29 Toxicogenomics and risk assessment<br>1.30 Functional annotation and novel transcripts<br>1.31 Conclusion<br>References<br><br>2. Pitfalls of transcriptomics and selection of the most appropriate transcriptomic technique<br>Xinmin Li, Ilya Belalov, and Anton Buzdin<br><br>2.1 Introduction<br>2.2 Pitfalls in specific transcriptomic techniques<br>2.3 Choosing the right tool for the job<br>2.4 Case studies and examples<br>2.5 Addressing pitfalls and challenges: Insights from recent research<br>2.6 Future directions in transcriptomics<br>2.7 Practical considerations and conclusion<br>References<br>Further reading<br><br>3. Bulk RNA sequencing in wet lab<br>Anton Buzdin, Ilya Belalov, Maria Suntsova, and Xinmin Li<br><br>3.1 Introduction to bulk RNA sequencing<br>3.2 Fundamental principles of bulk RNA sequencing<br>3.3 Key technologies and methodologies in bulk RNA sequencing<br>3.4 Sample collection and storage<br>3.5 RNA extraction and purification<br>3.6 Quality control measures<br>3.7 Library preparation for bulk RNA sequencing<br>3.8 Early barcoding and cost efficiency<br>3.9 Direct RNA isolation strategies<br>3.10 Sequencing platforms and technologies<br>3.11 RNA sequencing protocols<br>3.12 Overview of advanced RNA sequencing protocols<br>3.13 Reagents and equipment for bulk RNA sequencing<br>3.14 Technical and analytical quality control in RNA sequencing<br>3.15 Bioinformatics and data analysis<br>3.16 Data preprocessing and normalization methods for bulk RNA sequencing<br>3.17 Sequencing depth and coverage in bulk RNA sequencing<br>3.18 Differential expression analysis in RNA sequencing<br>3.19 Functional genomics and pathway analysis in RNA sequencing<br>3.20 Emerging applications of bulk RNA sequencing in disease research, immunology, and oncology<br>3.21 Challenges and limitations of current RNA sequencing methodologies<br>3.22 Future directions and technologies in RNA sequencing<br>3.23 Best practices for bulk RNA sequencing in the wet lab<br>3.24 Ethical considerations in transcriptomics research<br>3.25 Conclusion<br>References<br><br>4. Single-cell RNA sequencing in wet lab<br>Anton Buzdin, Xinmin Li, Maria Suntsova, and Ilya Belalov<br><br>4.1 Overview of what scRNA-seq is and why it is indispensable in research<br>4.2 Types of scRNA-seq protocols<br>4.3 Reagents and materials<br>4.4 scRNA-seq platforms: Comparison of chromium 10X, Drop-seq, and Smart-seq<br>4.5 Sample preparation and handling<br>4.6 Single-cell isolation techniques<br>4.7 Library preparation<br>4.8 Sequencing technologies<br>4.9 Techniques to ensure the integrity and quality of scRNA-seq data<br>4.10 Data analysis methods to validate the quality of sequencing results<br>4.11 Recommended sequencing depths for different types of analyses<br>4.12 Overview of computational tools and methods for analyzing scRNA-seq data<br>4.13 Integrating scRNA-seq data with other data types<br>4.14 Research and clinical applications of scRNA-seq<br>4.15 Innovations and future trends in scRNA-seq technology<br>References<br>Further reading<br><br>5. Spatial transcriptomics<br>Ilya Belalov, Ye Wang, Anton Buzdin, and Xinmin Li<br><br>5.1 Introduction<br>5.2 Spatial transcriptomics technologies<br>5.3 Imaging-based technologies<br>5.4 Sequencing-based technologies<br>5.5 Applications for basic, translational, and clinical research<br>5.6 Considerations for a first ST experiment<br>5.7 Concluding remarks<br>References<br><br>6. High-throughput and multiplex PCR in wet laboratory to measure gene expression<br>Anton Buzdin, Ilya Belalov, and Galina Zakharova<br><br>6.1 Introduction<br>6.2 Importance of high-throughput and multiplex PCR<br>6.3 Protocols for high-throughput and multiplex PCR<br>6.4 Reagents and requirements for material<br>6.5 Material requirements<br>6.6 Technical and analytical quality control (QC)<br>6.7 Analytical QC<br>6.8 Dynamic intervals and sensitivity<br>6.9 Sensitivity and specificity<br>6.10 Applications and sequencing depth recommendations<br>6.11 Recommended sequencing depths<br>6.12 Key studies and contributions<br>6.13 Further reading<br>6.14 Future directions and innovations<br>6.15 Challenges and limitations<br>6.16 Conclusion<br>References<br><br>7. Processing primary gene expression data: Normalization, harmonization and data quality control<br>Nicolas Borisov, Maksim Sorokin, and Anton Buzdin<br><br>7.1 Background<br>7.2 Principles of harmonization algorithms<br>7.3 Evaluation of the quality of harmonization<br>7.4 Validation of Shambhala-1/2 protocol on bulk mRNA-seq profiles<br>7.5 Differential clustering of human normal and cancer expression profiles<br>7.6 Correlation, regression, and sign-change analysis of cancer drug balanced efficiency scores after application of different methods of harmonization<br>7.7 Retention of biological properties after uniformly shaped harmonization<br>7.8 Validation of Shambhala-1/2 protocol on sc-mRNA-seq profiles<br>7.9 Discussion<br>Abbreviations<br>References<br><br>8. Data check and differential gene analysis<br>Maksim Sorokin and Anton Buzdin<br><br>8.1 Primary RNA expression data analysis<br>8.2 Finding differentially expressed genes and gene sets<br>8.3 Concluding remarks<br>References<br><br>9. Molecular pathway analysis using transcriptomic data<br>Nicolas Borisov, Maksim Sorokin, Igor Kovalchuk, Marianna Zolotovskaia, and Anton Buzdin<br><br>9.1 Background<br>9.2 Topology-based methods for pathway activation assessment<br>9.3 Methods for database preparation for pathway activation assessment<br>9.4 Personalized ranking of cancer drugs based on pathway activation levels<br>9.5 Concluding remarks<br>Abbreviations<br>References<br><br>10. Omics wise analysis of RNA splicing<br>Alexander Modestov, Anton Buzdin, and Vladimir Prassolov<br><br>10.1 Introduction<br>10.2 Omics technologies for RNA splicing analysis<br>10.3 RNA splicing products and regulators as biomarkers and targets of therapy<br>10.4 Challenges and future prospects<br>References<br><br>11. Transcriptome-wide analysis of protein synthesis: Ribosome profiling and beyond<br>Sergey E. Dmitriev, Daniil Luppov, Leonid M. Kats, Aleksandra S. Anisimova, and Ilya M. Terenin<br><br>11.1 Introduction<br>11.2 Ribosome profiling quantifies gene expression at the translational level<br>11.3 Ribosome profiling deciphers cryptic coding potential of the genome<br>11.4 Analyses of ribosome positional distribution and transcriptome-wide metagene profile reveal new phenomena<br>11.5 Molecular mechanisms revealed by variants of ribosome profiling and relative methods<br>11.6 Technical challenges, limitations, and artifacts in ribosome profiling studies<br>11.7 Ribosome profiling of cultured mammalian cells: An example protocol<br>Acknowledgments<br>References<br>Further readings<br><br>12. Transcriptomic biomarkers in biomedicine<br>Anton Buzdin, Alf Giese, Xinmin Li, and Ye Wang<br><br>12.1 Theoretical background<br>12.2 Molecular pathway biomarkers in oncology<br>12.3 Other applications of pathway biomarker analysis in biomedicine<br>12.4 Conclusion<br>References<br><br>13. Translational transcriptomics for personalized oncology<br>Anton Buzdin, Alexander Seryakov, Marianna Zolotovskaia, Maksim Sorokin, Victor Tkachev, Alf Giese, Marina Sekacheva, Elena Poddubskaya, Julian Rozenberg, and Tharaa Mohammad<br><br>13.1 Transcriptomics in clinical oncology<br>13.2 Detection of oncogenic fusion events<br>13.3 Detection of pathogenic splicing or exon skipping events<br>13.4 Gene signatures<br>13.5 Algorithmic approach in personalized oncology<br>13.6 Molecular pathway approach for personalized prediction of cancer drug efficacy<br>13.7 Concluding remarks<br>References<br><br>14. Transcriptomics for modern biotechnology<br>Anton Buzdin, Denis Kuzmin, Andrew Garazha, and Ilya Belalov<br><br>14.1 Introduction<br>14.2 High-throughput transcriptomic pipelines<br>14.3 Oncobox pathway activation levels quantization<br>14.4 Connectivity map assay<br>14.5 Databases and software for transcriptomic analysis<br>14.6 Industrial and startup landscape<br>14.7 Case studies and recent advances<br>14.8 Conclusion<br>References<br><br>15. Transcriptomics and quantitative proteomics: Competition or symbiosis?<br>Anton Buzdin, Sergey Moshkovskii, Xinmin Li, and Ilya Belalov<br><br>15.1 Introduction<br>15.2 Rationale for direct protein level measurement<br>15.3 Current progress in quantitative proteomics<br>15.4 Quality metrics comparison<br>15.5 Gene products interrogated<br>15.6 Integration of multi-omics data<br>15.7 Future perspectives<br>15.8 Conclusion<br>References<br><br>16. Quality assessment of differentially expressed gene signatures<br>Alexey Stupnikov and Anton Buzdin<br><br>16.1 Introduction<br>16.2 Conclusions<br>Acknowledgments<br>References<br><br>17. Detection of fusion transcripts by RNA-sequencing data<br>Ivan Musatov, Maksim Sorokin, and Anton Buzdin<br><br>17.1 Introduction<br>17.2 Basic software methods for detecting fusion genes<br>17.3 Algorithms based on alignment. STAR and STAR-Fusion<br>17.4 LongGF<br>17.5 Pros and cons of existing tools for bioinformatic fusion detection<br>17.6 Prospects for the development of tools and approaches to search for fusion transcripts<br>17.7 Conclusion<br>References<br><br>Index</p>