How Our Services Work

RNA sample submission

High-quality samples are important for accurate RNA sequencing. At the initial consultation, we offer recommendations on suitable extraction and clean-up methods. Alternatively, you can take advantage of our expertise and submit your samples to our RNA isolation service.

mRNA or whole transcriptome
We recommend that you send us 260 ng total RNA, but we get high-quality results with as little as 100 ng total RNA + 60 ng for QC.

Ultra-low mRNA
For ultra-low mRNA NGS, RNA isolation is included in the project and must be performed by us as we have developed an optimized NGS workflow with proprietary steps including unique RNA isolation and QC protocols specifically optimized for samples with low RNA content. Ultra-low mRNA sequencing is ideal for small amounts of cells or tissue including fine needle biopsies, LCM samples and sorted cells. We recommend that you send us a minimum of 100 cells to get high-quality results.

Biofluid miRNA
For miRNA-seq in serum and plasma, RNA isolation is included in the project and has to be performed by us. Our optimized NGS workflow with proprietary steps, including unique RNA isolation and QC protocols, is specifically optimized for miRNA sequencing from serum/plasma. Although we get high-quality results from as little as 250 µl, we recommend sending 500 µl of serum/plasma. We accept human serum/plasma samples, and samples from other species or other biofluids (urine, CSF, saliva, etc.) upon request.

Sample preparation

We offer to isolate RNA from your samples for sequencing. We perform high-quality RNA isolation from a range of sample types including cells, tissues, FFPE sections and blood. We also perform RNA isolation from minute amounts of sample as part of our Ultra-low mRNA Sequencing Service.

Biofluid miRNA
Isolation of RNA from biofluids is performed using QIAGEN kits and protocols developed specifically for this sample type.

RNA sample quality control (QC)
After receiving your RNA samples, we will determine the integrity and quantity of each sample using a Bioanalyzer/TapeStation and Nanodrop instruments and possible contaminations are assessed for each sample. You will receive a report with the results of these analyses prior to the library preparation and sequencing. We also perform the QC prior to any RT-reaction for downstream qPCR profiling.

Ultra-low mRNA
Following RNA isolation, we will perform RNA QC using qPCR-based methods optimized specifically for low RNA content samples. The RNA input amount is normalized by quantification of GAPDH and ß-actin using qPCR. You will receive a report with the results of these analyses prior to the library preparation and sequencing.

Biofluid miRNA 
For biofluid samples, QC of the RNA is performed by qPCR, using a unique combination of RNA spike-ins and endogenous miRNAs to detect outlier samples and monitor RNases, RNA isolation efficiency, hemolysis and presence of inhibitors that may affect library preparation. You will receive a report with the RNA-QC results prior to the library preparation and sequencing.

Library preparation

After QC, we design libraries according to the type of sequencing to be performed. For mRNA sequencing, we enrich poly-A mRNA molecules from total RNA using an Oligo-dT magnetic bead-based system. For whole transcriptome sequencing, we perform ribosomal RNA depletion (through the use of a biotin-streptavidin bead-based system). The resulting RNA is fragmented and converted into mRNA or whole transcriptome cDNA NGS libraries. A Bioanalyzer/TapeStation is used to perform QC of the libraries.

Biofluid miRNA 
We generate libraries and select size using a bead-based size selection of 15-40 nt. Library QC is performed using a Bioanalyzer/TapeStation.

Sequencing

We offer mRNA and whole transcriptome sequencing using the Illumina platform. We use both NextSeq 500 and HiSeq 2500 instruments, enabling us to cater for a range of project sizes. For standard projects, the sequencing parameters are 1 x 75 bp, 30 M reads per sample or 2 x 75 bp, 60 M paired-end reads per sample. However, the sequencing parameters can be tailored to your specific requirements. Please contact us for further information.

miRNA
We offer 2 types of miRNA sequencing using the Illumina platform:

• miRNA sequencing including miRNA discovery
• Biofluid miRNA sequencing including miRNA discovery

Sequencing is performed using Illumina instruments, 1x 75bp reads, 10M reads per sample.

Biofluid miRNA
Biofluid miRNA sequencing is performed using Illumina instruments, 1x 75bp reads, 10M reads per sample.

Analysis and interpretation for mRNA and whole transcriptome NGS projects

Comprehensive data analysis, including statistical analysis is provided using our unique NGS data analysis pipeline:

• Comprehensive QC of raw data
• Mapping: Alignment of sequence to the appropriate reference genome
• QC of mapped data
• Quantification of known transcripts (both Ensembl and UCSC are supported)
• Prediction and quantification of novel transcripts
• Tests for significant changes in promotor usage
• Identification of splice-junctions with list of exon positions associated with each transcript
• Identification of alternative splicing and splice variants with list of exons associated with each isoform
• Identification of antisense transcripts
• Normalization
• Differential gene expression analysis to identify known and novel genes and transcripts that are differentially expressed between your sample groups.
• Statistical analysis is performed, provided there are a sufficient number of samples per group
• Unsupervised analysis: Principal Component Analysis (PCA plot) and heat map   
• Supervised analysis: For each specific group comparison we generate a Volcano plot to display genes which are differentially expressed between the groups, list differentially expressed genes as well as identify statistically significant signals
• Gene Ontology (GO) enrichment analysis is performed to identify GO terms that are over-represented within differentially expressed genes. This gives a picture of the impact of the experiment on biological pathways and processes.

Biofluid miRNA

• Mapping to miRBase and identification of predicted novel miRNAs
• IsomiR counts can be reported
• For biofluids: QC includes analysis of >50 spike-ins which are used to check the reproducibility and linearity of the sequencing data
• Mapping sequences to the appropriate reference genome
• In connection with large service projects, we also offer fully extensive customized bioinformatics analysis
• Normalization
• Differential gene expression analysis to identify known and novel genes and transcripts that are differentially expressed between your sample groups.
• Statistical analysis is performed, provided there are a sufficient number of samples per group
• Unsupervised analysis: Principal Component Analysis (PCA plot) and heat map   
• Supervised analysis: For each specific group comparison we generate a Volcano plot to display genes which are differentially expressed between the groups, list differentially expressed genes as well as identify statistically significant signals
• Gene Ontology (GO) enrichment analysis is performed to identify GO terms that are over-represented within differentially expressed genes. This gives a picture of the impact of the experiment on biological pathways and processes.

Report and final consultation

The final report is delivered by our cloud-solution called XploreRNA and contains:

• An easy-to-read data report containing a description of the project, assessments of sample and data quality and an overview of the results of the data analysis with publication-grade illustrations   
• A data analysis file which is sufficient for publication, or performing your own analysis of the data if needed   
• A materials and methods section ready to use for publication purposes   
• The complete encrypted raw data (including FASTQ and BAM files) are provided

Turnaround times
mRNA or whole transcriptome and miRNA: 8–10 weeks

Ultra-low mRNA amounts or biofluids: 8–10 weeks

After you have received your report, we arrange a follow-up discussion to answer any questions you may have about your report and discuss the significance of your results and next steps. We can also help you design appropriate miRCURY LNA or RT2 qPCR assays to validate your NGS data.