mRNA-Seq

CD Genomics's mRNA-Seq service, powered by the state-of-the-art Illumina NovaSeq platforms, offers comprehensive solutions for gene expression quantification, differential gene expression analysis, identification of novel transcript isoforms, alternative splicing, and gene fusions, etc. Our expert team works closely with you to provide standard and customized high-throughput sequencing and bioinformatics analyses based on project-specific needs, as well as exquisite results for publication.

Overview

Messenger RNA (mRNA) is one type of RNA transcripts that corresponds to the genetic sequence of a gene, instructing the protein synthesis in cells. Eukaryotic mRNA sequencing (mRNA-Seq), by leveraging the technology of next-generation sequencing (NGS), reveals the expression profiles of mRNA in a given biological sample, as well as differential gene expression among sample groups. The mRNA-seq provides a clear, complete, high-resolution view of the coding transcriptome, including detection of known and novel transcript isoforms, identification of gene fusion events and other features, as well as gene expression analysis by measuring transcript abundance. In contrast with whole transcriptome sequencing that sequences all coding and non-coding transcripts, mRNA-seq focuses on the coding RNAs, which are around 2% of the whole transcriptome and contain a poly-A tail. The mRNA-seq delivers a comprehensive picture of the coding transcriptome without the need of prior knowledge and is advantageous over gene expression arrays and other approaches. mRNA-seq has been widely used for identification and comparison of gene expression levels between groups, perfect for drug treatment, behavioral selection, identification of cellular responses to viruses, and disease related research.

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Data Quality Control
• De novo assembly or reference assembly
• Gene (including oncogene) functional annotation
• Gene expression quantification
• Differential expression profiling
• GO and KEGG enrichment analyses
• Protein-Protein Interaction (PPI) analysis
• Transcription factors prediction
• SNP & InDel detection
• Alternative splicing analysis
• Fusion gene prediction

Sample Requirements

• RNA sample (quantity ≥ 1 μg, concentration ≥ 300 ng/μL)
• RIN ≥ 6.8 with smooth base line; 28S/18S ≥ 1.5; OD260/280 =1.8-2.2; OD260/230: ≥ 1.8.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: raw data as BAM files, coverage summary, QC report, custom bioinformatics analyses.

Demo Results

FAQ

• • What are the requirements for blood samples to do RNA-seq?

    • Blood samples for RNA extraction require the removal of white blood cells. The concentration of RNA in the blood is low and requires 2mL of sample and about 3uL of total RNA extracted.

• • What is the recommended data volume for eukaryotic mRNA-seq?

    • Sequencing mode: 2×100 (150) bp, data volume: 20~30M reads/sample.

• • Why is mRNA sequencing recommended compared to microarray expression profiling?

    • Transcriptome measures complete transcript information, while expression profiling measures partial cDNA sequences; compared with gene expression microarray, transcriptome not only compares differential expression, but also predicts new transcripts and variable splicing; transcript sequencing can be done without a reference genome, while microarrays must rely on a reference genome.

• • What are FPKM and RPKM?

    • FPKM and RPKM are used to calculate gene expression levels. FPKM is indicating the number of fragments from a particular gene per Kilobase of transcript sequence per Millions base pairs sequenced. RPKM is indicating the number of reads per Kilobase of transcript sequence per Millions base pairs sequenced from a particular gene. It can be seen that FPKM is in terms of fragment (read pair), while RPKM is in terms of read (single read). In addition, we used FPKM to quantify the transcripts of genes.

References:

1. Wahlstedt H, Daniel C, Ensterö M, et al. Large-scale mRNA sequencing determines global regulation of RNA editing during brain development. Genome research, 2009, 19(6): 978-986.
2. Park B J, Ahn H S, Han S H, et al. Analysis of the Immune Responses in the Ileum of Gnotobiotic Pigs Infected with the Recombinant GII. p12_GII. 3 Human Norovirus by mRNA Sequencing. Viruses, 2021, 13(1): 92.

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