DNA AND RNA QUALIFICATION
ABOUT
The quality/integrity of DNA or RNA greatly affects experimental outcome of any downstream application. A couple of examples :
-
Highly degraded DNA is not suitable for e.g. long range PCR, this as the primers can't anneal to an intact DNA fragment
-
Highly degraded RNA is not suitable for poly-A based RNA-seq if you are interested in sequencing full transcripts, this as you will only enrich for the 3' (polyadenylated) ends of your transcripts
-
...
The technologies listed below provide you with a quality assessment of your biomolecule of interest.
Next to the quality, you might also be interested in the quantity/concentration of your samples. For this, use the button below.
METHODOLOGY
The integrity of biomolecules (DNA, RNA and protein) can be analyzed through gel electrophoresis, which in an automated systems is referred to as either (1) capillary electrophoresis or (2) microchip electrophoresis.
In essence, the biomolecules are seperated over a polymer in a (micro)capillary by applying a current over this capillary. This way, the biomolecules are (1) separated by size as the polymer acts as a sieve (larger fragments migrate slower). But the molecules are also (2) separated by charge. The biomolecules are detected when they pass by a detection window. There, a laser excites the dye attached to the biomolecules of interest. These biomolecule-specific dyes are pre-loaded in the polymer for convenience.
By utilizing internal markers (lower and upper markers) and cross-comparing to an external molecular ladder, an accurate size determination (sizing) is possible.
As capillaries are much more resilient to higher currents when compared to slab gels, separation times are highly reduced : ranging from ca. 30s to ca. 1h dependent on device and application.
Also, as the devices analyze the samples directly from a multititerplate (96 or 348 well plates), there's no need to load samples manually on a gel.
The technology for (1) capillary electrophoresis or (2) microchip electrophoresis is highlighted in the videos below.
AATI/Agilent Fragment Analyzer : example of capillary electrophoresis
Caliper/Perkin Elmer LabChip : example of microchip electrophoresis
INSTRUMENTS
The following devices are used for DNA/RNA qualification in our premises.
AATI Fragment Analyzer
Capillary electrophoresis device that enables qualification and quantification of (genomic) DNA samples, but also RNA samples.
Caliper LabChip GX Touch
Microchip electrophoresis device that enables speedy qualification of DNA samples of up to 6Kb in size.
SPECIFICATIONS
The specifications are subdivided in 2 sections : (1) the assay metrics and (2) applications & remarks.
More information can be found on the supplier page through the corresponding buttons in the 'Assay metrics' section.
Assay metrics
Instrument
Sample type
Kits currently in use
Sensitivity
Run time
Size range
* Run times are for the installed 33 cm capillary array. Before each run, the capillaries need to be conditioned (takes ca. 15 min)
** Before each run, the microfluidics chip needs to be primed and heated. This takes approximately 15 minutes before the start of a run.
Applications & remarks
Instrument
Applications (possible)
Applications (in use)
Remarks
AATI Fragment Analyzer
DNA/NGS fragment analysis, Genomic DNA analysis, Large Fragment analysis, RNA analysis, RQN/RIN calculation, GQN calculation, DV200 calculation, Single stranded DNA (ssDNA) analysis, Cas9 mRNA polyadenylation QC, Estimate CRISPR homology-directed repair (HDR) efficiency
DNA/NGS fragment analysis, Genomic DNA analysis, Large Fragment analysis, RNA analysis, RQN/RIN calculation, DV200 calculation
The Fragment Analyzer is a versatile device allowing for 2 simultaneous assays to run (e.g. an RNA and NGS assay) with a superior sensitivity or limit of detection.
This device uses only 2 µl of the native sample to be diluted with 'Diluent marker' solution.
Using a 12-capillary array, the run-time per sample is quite high.
A total of 3 plates (96 well only) can be loaded simultaneously.
The data analyis software is less convenient as compared to the LabChip software.
Caliper LabChip GX Touch
DNA/NGS fragment analysis, Genomic DNA analysis, Large Fragment analysis, RNA analysis, RQN/RIN calculation, GQN calculation, DV200 calculation, Estimate CRISPR homology-directed repair (HDR) efficiency
DNA/NGS fragment analysis
The LabChip is unmatched for its speed and ability to work with the native sample (no mixing of sample with 'diluent marker' needed) from either a 96 well or 384 well plate. However, as the device needs a volume of (minimally) 25 µl, we mostly need to dilute the sample with nuclease free water beforehand.
As this device can not easily switch between different assays, current assays are limited to DNA-based assays.
Used abbreviations : RQN = RNA Quality Number ; RIN = RNA Integrity Number ; GQN = Genomic DNA Quality Number ; DV200 = % of RNA > 200 nt
CERTIFICATES
Part of accredited process
DOWNLOADS
Fragment Analyzer QC report
1
CONTACTS
BRIGHTcore general inquires
+32 (0)2 477 64 79 (sec)
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 us.
Tissue | Recipient | Quantity | Transport | Purpose / Extract |
|---|---|---|---|---|
DNA from various tissues | Microtube* or 96 well plate | See test details/specifications | Room temperature | DNA |
NGS library (PCR free) | Microtube* or 96 well plate | See test details/specifications | Ice (max. 1 day) or dry ice | DNA |
NGS library (non-PCR free) | Microtube* or 96 well plate | See test details/specifications | Room temperature or ice pack | DNA |
PCR products (amplicons) | Microtube* or 96 well plate | See test details/specifications | Room temperature | DNA |
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 |
* 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 contact us.
Deliverable | Description | Research or diagnostic |
|---|---|---|
Fragment analysis - pdf | Summary pdf report with DNA/RNA electrophoresis results (see downloads) | RUO (on request) |
Raw data - raw file (folder) | The raw data output of the AATI/Agilent Fragment Analyzer devices | RUO (on request) |
Raw data - gxd file | The raw data output of the Caliper/Perkin Elmer LabChip devices | RUO (on request) |
PLAN EXPERIMENT
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 :
-
What type of sample would you like to analyze?
-
Which elution buffer did you use?
-
What are the expected concentrations of your samples?
-
How many samples would you like to process?
-
How fast do you need the results (TAT) and/or do we need to expedite?
-
If you deviate from our sample types, are there test samples available?
-
Are our standard deliverables ok?
-
...
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 :
-
Coordinates of the person to whom to address the quotation
-
Which type of test you'd like to set up
-
Do you require a specific deliverable?
-
Do we need to expedite (comes at extra cost)?
-
...
2
Submit your samples
Once you agree with our terms (quote / TAT / terms & conditions) :
-
Fill out our sample submission form
-
Send it via email : contact us
-
Print out the form and include it with your samples
-
-
Send your samples to us
-
Follow our device dependent instructions : see details here
-
Label your tubes/plates correctly : see details here
-
Adhere to the corresponding transport conditions listed under Sample types
-
-
Indicate if we can discard your samples after completion of project
3
Your experiment starts
Now it is up to us... We will start your experiment as soon as possible.
-
When expedited, the turn-around-time (TAT) is 2 workdays
-
For standard requests the turn-around-time (TAT) is 5 workdays
The standard 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.
