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mRNA Sequencing (mRNA-seq)

Introduction to RNA Sequencing

RNA sequencing (RNA-seq) has been transforming the study of cellular functionality, which provides researchers with an unprecedented insight into the transcriptional landscape of cells. Employing the high-throughput and accurate next-generation sequencing technique (NGS), RNA-seq reveals gene expression profiles and describes the continuous variations in the transcriptome. In the RNA-seq technique, the single-stranded messenger RNAs (mRNAs) are selectively captured or enriched, and converted to complementary DNA (cDNA) for library preparation.

At Novogene, the state-of-the-art Illumina NovaSeq platforms are used for a paired-end 150 bp sequencing strategy (short-reads) to sequence the cDNA libraries. With strong sequencing capacity and rich experience, Novogene offers multiple services to meet various needs, including the access to not only eukaryotic mRNA sequencing (mRNA-seq) services, but also the studies on prokaryotic transcripts, non-coding RNAs, full-length isoforms (long-reads), whole transcriptomes, and meta-transcriptomes.

How does mRNA Sequencing work?

mRNA-seq is a powerful tool to analyze the cell transcriptome profile. Novogene’s professional services help on research goals in a wide range of applications, including:

  • Quantitative profiling of transcripts in different tissues or samples, under various conditions and treatments
  • Discovery of novel transcripts, alternative splicing (AS), and transcript variations
  • Research of developmental mechanisms and drug resistance through tissue-specific transcripts or time-course gene expression
  • Biomarker discovery based on novel transcripts/isoforms, SNP/InDel identification, and fusion gene analysis
  • Omics analysis in combination with the transcriptome
  • Investigation of the pathogenic mechanism and clinical subtypes in clinical diagnosis

Benefits of Novogene mRNA Sequencing

Novogene mRNA-seq offers high throughput and high accuracy (with Q30 score ≥ 80%) coupled to low initial RNA amounts required. Novogene has extensive experience providing RNA-Seq services, having successfully completed thousands of projects and helped multiple researchers to publish in high Impact Factor journals.

Novogene offers inclusive solutions for quantification, differential gene expression, annotation of novel transcripts, alternative splicing, discovery of fusion gene, and other potential variations. Highly-qualified bioinformaticians deliver and publication-ready analysis results using personalized pipelines for species either with or without a reference genome.

mRNA-seq Specifications: Sample Requirements

Library Type Sample Type Amount RNA Integrity Number (Agilent 2100) Purity (NanoDrop)
Eukaryotic RNA-Seq (cDNA library) Total RNA ≥ 0.4 μg ≥ 6.8 (Animal), with smooth base line
≥ 6.3 (Plant and Fungus), with smooth base line
OD260/280 = 1.8-2.2
OD260/230 ≥ 1.8
Total RNA (Blood) ≥ 0.8 μg
Amplified cDNA (double-stranded) ≥ 100 ng Fragments between 400bp and 5000bp with main peak at ~2000bp OD260/280 = 1.8-2.0
OD260/230 ≥ 1.8
Eukaryotic RNA-Seq (strand specific library) Total RNA ≥ 0.8 μg ≥ 6.8 (Animal), with smooth base line
≥ 6.3 (Plant and Fungus), with smooth base line
OD260/280 = 1.8-2.2
OD260/230 ≥ 1.8

Note: Sample amounts are listed for reference only. Download the Service Specifications or Sample Requirements to learn more. For detailed information, please contact us with your customized requests.

mRNA-seq Specifications: Sequencing and Analysis

Sequencing Platform Illumina NovaSeq 6000 Sequencing System
Read Length Paired-end 150 bp
Data Output
  • ≥ 20 million read pairs per sample for species with reference genome
  • ≥ 50 million read pairs per sample for species without reference genome (de novo transcriptome assembly projects)
  • Data Analysis Capability
  • Data Quality Control
  • Gene expression quantification
  • Differential expression profiling
  • Functional enrichment analysis
  • Novel transcripts identification
  • SNP & InDel analysis
  • Alternative splicing (AS) analysis
  • Fusion gene prediction
  • Protein-Protein Interaction (PPI) analysis
  • Transcription factors and oncogene functional annotation
  • Note: Recommended data outputs and analysis contents displayed are for reference only. Download the Service Specifications to learn more. For detailed information, please contact us with your customized requests.

    Project Workflow of Novogene mRNA-seq Services

    The project workflow starts with sample quality control (Sample QC) to ensure that your samples meet the criteria of the RNA-Seq technique. Then, the appropriate library is prepared according to your target organism and application, and subsequently tested for its quality (Library QC). Next, a paired-end 150 bp sequencing strategy is used to sequence the samples and the resulting data is also check for its quality (Data QC). Finally, bioinformatic analyses are performed and publication-ready results are provided.The following flowsheet describes the step-by-step protocol our mRNA-seq technique follows.

    Preparation of sample is followed by the RNA library preparation. RNA library is formed by ployA caputure (or rRNA removal) and reverse transcription of cDNA. Illumina PE150 technology is employed to sequence the sample and the final stage involves the bioinformatics analysis.

    Featured Publications using Novogene’s RNA-seq service

    RNA-seq (mRNA-seq) is the most frequently cited NGS method. Here we have summarized some outstanding academic publications that used Novogene RNA sequencing (mRNA Sequencing) services.

    Error Rate Distribution

    error rate for Novogene RNA-seq

    The x-axis shows the base position along each sequencing read and the y-axis shows the base error rate.

    GC Content Distribution

    GC Content Distribution for Novogene RNA-seq

    Horizontal axis for reads position, vertical axis for single base percentage. Different color for different base type.

    Classification of Raw Reads

    Classification of Raw Reads for Novogene RNA-seq

    Reads Distribution on Reference Genome

    Reads Distribution on Reference Genome for Novogene RNA-seq

    Gene Expression Quantification

    Gene Expression Quantification for Novogene RNA-seq

    X axis represents the name of sample, Y axis indicates the log10(FPKM+1), parameters of box plots are indicated, including maximum, upper quartile, mid-value, lower quartile and minimum.

    Volcano Plot of changes on Gene Expression

     Volcano Plot of changes on Gene Expression for Novogene RNA-seq

    Horizontal axis for the fold change of genes in different samples. Vertical axis for statistically significant degree of changes in gene expression levels, the smaller the corrected pvalue, the bigger -log10(corrected pvalue), the more significant the difference. The point represents gene, blue dots indicate no significant difference in genes, red dots indicate upregulated differential expression genes, green dots indicate downregulated differential expression genes.

    Hierarchical Clustering Heatmap of Differential Expression

    Hierarchical Clustering Heatmap of Differential Expression for Novogene RNA-seq

    The overall results of FPKM cluster analysis, clustered using the log10(FPKM+1) value. Red denotes genes with high expression levels, and blue denotes genes with low expression levels. The color ranging from red to blue indicates log10(FPKM+1) value from large to small.