RNA SEQUENCING
ABOUT
Via RNA sequencing (RNAseq), it is possible to infer the transcriptome of your cells or tissue of interest. So via transcriptomics or expression profiling, it is possible to detect or quantitate (relatively) a whole array of RNA species, ranging from small RNAs (e.g. micro RNA = miRNA) to messenger RNA (mRNA) and long non-coding RNA (lncRNA).
The methodology selected for RNAseq is greatly determined by the quality and quantity of your input material, but also the desired output. More information about this can be found in the 'Methodology' section or in the discussion of the individual applications.
For more information about quantifying and qualifying RNA samples, please consult the corresponding chapters.
METHODOLOGY
The principle of RNAseq relies on the ability to generate a high quality cDNA bank (without 'bulk' RNA, like ribosomal RNA or globin RNA), followed by a DNA library preparation & sequencing. The last 2 topics are addressed in the following chapters.
A large amount of RNAseq options & methodologies exist. In order to get to the correct choice you need to consider the following aspects :
Which output would I like?
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When you need small RNAs (smRNA) or micro RNAs (miRNA), this would be dedicated assay where you lose information on all other (longer) RNA species.
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When you require long non-coding RNA (lncRNA), it might be best to chose a ribodepletion based library prep in order to profile both the polyadenylated as non-polyadenylated lncRNA fragments.
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When you require coding (polyadenylated) full transcripts, both a ribodepletion as poly-A based library prep is possible
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When you only require the 3' end sequences of coding (polyadenylated) RNA, we require a specific/dedicated assay and you lose information of any splice variation and non-polyadenylated fragments.
What quantity of material do I have?
Generally, the more input the better. At (ultra-) low inputs you should anticipate stochastic effects and dropouts. When analyzing ultra-low inputs, data analysis is commonly restricted to high expressing genes. For inputs <10 ng RNA, we work with single-cell applications.
At low inputs, poly-A based library preps are more frequently used as compared to ribodepletion methods.
What quality of material do I have?
Working with high quality RNA (RIN/RQN >7) enables the analysis of full transcripts, whatever the method you select (poly-A / depletion based). However, for degraded RNA - like RNA derived from FFPE tissue - it is only possible to sequence the full length transcripts when using a ribodepletion based methodology, where the cDNA synthesis is performed using random hexamers or the combination of random hexamers and poly-dT primers.
A matrix of our current RNAseq methods and their applications/abilities are listed below. For more information, please consult the relevant application pages.
Reads
Method
mRNA
lncRNA
Full transcripts
3' ends only
Stranded reads
Poly-A based
Ribodepletion
Degraded RNA
Kit
miRNA
RNA species
Quality requirement
Our input
Input range
- : not applicable / not detectable
+ : applicable / detectable
+/- : partly applicable / partly detectable
INSTRUMENTS
The following devices are used for RNA sequencing in our premises.
Illumina NovaSeq 6000
Our highest capacity short read sequencer. This device can generate reads up to 250 bp in length (on the SP flowcell) and generate up to 10B reads per flowcell (S4).
CERTIFICATES
Under validation
DOWNLOADS
CONTACTS
BRIGHTcore general inquires
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UZ Brussel
BRIGHTcore
Laarbeeklaan 101
1090 Brussels
SAMPLE TYPES
We aim to keep the list with sample types updated. However, if you believe we offer this test on other sample types, or if you have a very specific sample type you'd like to evaluate : please contact the corresponding persons for either diagnostic or research purposes.
Tissue | Recipient | Quantity | Transport | Purpose / Extract |
|---|---|---|---|---|
Blood | Streck RNA Complete BCT | 10 ml | Room temperature | cfRNA/exoRNA |
Blood | PAXgene Blood RNA tube | 10 ml (2,5 ml blood) | Ice pack | RNA |
Cells of various tissue origins | Microtube* with cells on lysis buffer | > 5 million cells | Dry ice | RNA |
FFPE block of various tissues | Paraffin block (in cassette) | Representative block ; Tumor load preferentially >10% ; To be processed by our pathology department (microtome section needed) | Room temperature | RNA |
FFPE section of various tissues | Microtube* | Min. 1x 10 µm section (up to 2 mm³ tissue) ; Tumor load preferentially >10% | Room temperature | RNA |
Plasma | DNA LoBind 5 ml tube | > 2ml | Dry ice | cfRNA/exoRNA |
RNA from various tissues | Microtube* or 96 well plate | See test details/specifications | Ice (max. 20 min) or dry ice | RNA |
RNA from various tissues | Microtube* | See test details/specifications | Ice (max. 20 min) or dry ice | RNA |
Tissue of various origins | Cryovial containing stabilizer (eg. RNAlater) or tissue homogenized on lysis buffer | Application/tissue specific (contact us) | Dry ice | RNA |
* Microtube can be a either a cryovial or eppendorf tube (0,5 ; 1,5 or 2 ml). Please consider the quality of the tube used (nuclease free ; free of RNA/DNA ; sterile ; LoBind) according to application needed.
DELIVERABLES
Below, you can find the type of data files that can be retrieved for this test.
If you require more information or need a more custom output, please consult our Bioinformatics page.
Deliverable | Description | Research or diagnostic |
|---|---|---|
Run folder | The complete run folder can be transferred if it concerns a run reservation (private run) | RUO (on request) |
Raw data - fast5 file | Raw data file generated by Oxford NanoPore sequencers | RUO (on request) |
Raw data - bam file | Binary data file containing the aligned reads | RUO (on request) |
Raw data - fastq file | Data file containing the raw (unaligned) reads | RUO (on request) |
PLAN EXPERIMENT
Please get clearance from your local ethical committee and/or register your samples with a biobank.
To set experiments up with us, please follow the steps below.
Get in touch (ONLY FOR NEW PROJECTS)
Explain your project, so we can assist you to find the best solution.
Expect questions like :
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What type of sample would you like to analyze?
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Will you handle the RNA extraction (please know : BRIGHTcore doesn't handle human pathogens)?
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In which elution buffer did you elute your RNA (we prefer nuclease free water)?
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How many samples would you like to process?
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How fast do you need the results (TAT) and/or do we need to expedite?
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If you deviate from our sample types, are there test samples available?
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Do you require bioinformatics support or are our standard deliverables ok?
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...
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Request quotation
Once you know which experiment you want to set up, fill out the 'Quote request' form, with the essential details of your project :
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Coordinates of the person to whom to address the quotation
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Which type of test you'd like to set up
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Do you require bioinformatics support?
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Do we need to expedite (comes at extra cost)?
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...
2
Submit your samples
Once you agree with our terms (quote / TAT / terms & conditions) :
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Fill out our sample submission form
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Send it via email : contact us
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Print out the form and include it with your samples
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Send your samples to us
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Adhere to the corresponding transport conditions listed under Sample types
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Label your tubes/plates correctly : see details here
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Indicate if we can discard your samples after completion of project
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Your experiment starts
Now it is up to us... We will start your experiment as soon as possible. When expedited, we will start the experiment within 1 week. For standard requests the turn-around-time (TAT) is 2 months.
Please consider : we require minimally 1 week for library preparation and sequencing and 1 week for the standard Bio-IT output and data transfer.
Our TAT is generally overestimated, but this extended timeframe allows us to : combine similar requests of multiple scientists and to repeat the experiment in case of issues.
Please refrain from contacting us in case the pre-agreed TAT didn't expire yet.
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APPLICATIONS
