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?
  • 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.

  • 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.

  • When you require coding (polyadenylated) full transcripts, both a ribodepletion as poly-A based library prep is possible

  • 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 / depetion based). However, for degraded RNA - like RNA derived from FFPE tissue - it is only possible to sequence the full lenght transcripts when using a ribodeplection 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

Illumina TruSeq Stranded mRNA

-
+
+/-
+
-
+
+
-
-

RIN ≥ 8

100-1000 ng

200 ng

KAPA RNA HyperPrep Kit with RiboErase (HMR)

-
+
+
+
-
+
-
+
+

DV200 > 30%

25 -1000 ng

150 ng

Lexogen QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

-
+
+/-
-
+
+
+
-
+

DV200 > 30%

1-500 ng

150 ng

Takara SMART-Seq v4 Ultra Low Input RNA Kit

-
+
+/-
+
-
+
+
-
-

Intact RNA

0,01-10 ng

0,01-10 ng

Takara SMARTer smRNA-Seq Kit

+
+/-
-
+
-
+
+
-
+/-

RIN ≥ 8 (lower is possible)

1-2000 ng

500 ng

- : 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
Under validation

DOWNLOADS

 

CONTACTS

 
pas14klein.jpg

BRIGHTcore general inquires

+32 (0)2 477 64 79 (sec)

UZ Brussel

BRIGHTcore

Laarbeeklaan 101

1090 Brussels

SAMPLE TYPES

 

We aim to keep te list with sample types updated. However, if you believe we offer this test on other sample types, of if you have a very specific sample type you'd like to evaluate : please contact the corresponding persons for either diagnostic or research purposes.

arrow&amp;v
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 :

  1. What type of sample would you like to analyze?

  2. Will you handle the RNA extraction (please know : BRIGHTcore doesn't handle human pathogens)?

  3. In which elution buffer did you elute your RNA (we prefer nuclease free water)?

  4. How many samples would you like to process?

  5. How fast do you need the results (TAT) and/or do we need to expedite?

  6. If you deviate from our sample types, are there test samples available?

  7. Do you require bioinformatics support or are our standard deliverables ok?

  8. ...

Call Center Headset

1

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 :

  1. Coordinates of the person to whom to address the quotation

  2. Which type of test you'd like to set up

  3. Do you require bioinformatics support?

  4. Do we need to expedite (comes at extra cost)?

  5. ...

Image by Andre Taissin

2

Submit your samples

Once you agree with our terms (quote / TAT / terms & conditions)  :

  1. Fill out our sample submission form

    1. Send it via email : contact us

    2. Print out the form and include it with your samples 

  2. Send your samples to us

    1. Adhere to the corresponding transport conditions listed under Sample types

    2. Label your tubes/plates correctly : see details here

  3. Indicate if we can discard your samples​ after completion of project

Blood Samples

3

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.

Alarm Clock

4

APPLICATIONS

 

The following applications/kits are readily available for you to make use of. More info can be found on the dedicated pages.  If you don't find what you need, please contact us!