DNA AND RNA QUANTIFICATION
ABOUT
The quantity (and quality) of your DNA and RNA samples may greatly affect the outcome of your experiments & even affects the choice of the technology used. A couple of examples :
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When having ultra low DNA inputs, PCR-free library preparations are off the table
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Ultra high DNA (or RNA) inputs may lead to inhibition of certain enzymatic steps (e.g. fragmentation / cDNA synthesis)
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When having a high protein/aromatic compound/chaotropic salt/... contamination, one should contemplate on performing an addtional cleanup of the DNA/RNA sample in order to get a successful downstream experiment
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When setting up a new DNA or RNA extraction methodology, we advise to verify your sample both fluorometrically (dye-based) as spectrometrically (absorbance). This to assess both the yields as the potential contamination of your sample.
Once the extractions are a routine/controlled setup, we'd limit the quantifications to a fluorometric assessment.
Below, you can find the available instruments to quantify your biomolecule (DNA/RNA) of interest.
Next to the quantity/contamination, you might also be interested in the quality/integrity of your samples. For this, use the button below.
METHODOLOGY
The quantification of dsDNA and RNA at BRIGHTcore is routinely performed through the use of specific dyes, respectively (1) a double-stranded DNA-binding - intercalating - fluorescent dye and (2) a RNA-binding fluorescent dye. The advantages of a dye-based assay over an absorbance-based method are (1) the specificity for the biomolecule (dsDNA vs. RNA) to be quantified and (2) the sensitivity.
On the other hand, absorbance-based methods can - less accurately - quantify DNA and RNA by their absorbance at 260 nm, but also provide additional information like :
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A260/280 : indicative for protein, phenol or other aromatic compound contamination
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Normal range DNA : 1,7 - 1,8
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Normal range RNA : 1,8 - 2,0
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A260/230 : indicative for EDTA, carbohydrate, chaotropic salts (and also protein) contamination
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Normal range DNA and RNA : >1,8 (preferentially >2,0)
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The Perkin Elmer DropletQuant is a microfluidics absorbance based instrument (Trinean technology) that is able to discern absorbance profiles of DNA, RNA and contaminants thus allowing for accurate quantification of your biomolecule of interest.
Alternatively, we can also assist you on setting up qPCR based quantification experiments. As this requires a specific design for your target (and/or biomolecule) of interest, please contact us to explore the possibilities.
Invitrogen Qubit 2.0 : example of fluorometric assay
INSTRUMENTS
The following devices are used for DNA/RNA qualification in our premises.
Invitrogen Qubit 2.0
Basic fluorometer for single-tube concentration measurements of dsDNA, ssDNA, RNA and proteins
Perkin Elmer DropletQuant
High throughput UV-Vis spectrometer for the quantification of DNA, RNA and proteins
Perkin Elmer Victor Nivo 3F
High throughput fluorescence, luminescence and absorbance (filter based) plate reader.
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
Kits currently in use
Wavelengths
Run time
Sensitivity
* Sensitivity is calculated for our routine setup of 2 µl of sample + 198 µl of working solution
** Sensitivity is calculated for our routine setup of 2 µl of sample + 48 µl of working solution (1/2 volume plates)
Applications & remarks
Instrument
Applications (possible)
Applications (in use)
Remarks
Invitrogen Qubit 2.0
dsDNA quantification, ssDNA quantification, RNA quantification, Protein quantification
dsDNA quantification, RNA quantification
Extremely user-friendly device to quantify small batches of samples. The standard curve consists of a 2-points calibration.
Perkin Elmer DropletQuant
dsDNA quantification, ssDNA quantification, RNA quantification, Protein quantification, Absorbance measurement (spectrum), A260/A230, A260/A280
dsDNA quantification, A260/A230, A260/A280
DNA extracts are quantified on this device as results correlate nicely to our dye-based assays and additional info on contaminations (proteins, aromatic compounds,...) are rendered available through the A260/A280 and A260/A280 calculation. The full potential of this device is not yet unlocked due to absence of the complex data-analysis software (cDrop).
Perkin Elmer Victor Nivo 3F
dsDNA quantification, ssDNA quantification, RNA quantification, Protein quantification, Luminescence assays, Absorbance measurement (filter based), Fluorescence intensity measurement
dsDNA quantification
When quantifying large batches of samples (from 24 samples on) this device is preferred. Per batch (= per working solution formulation) a 12 point standard curve is calculated to ensure proper quantification.
CERTIFICATES
Part of accredited process
DOWNLOADS
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 |
* 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 |
|---|---|---|
Concentrations - Excel file | An MS Excel file (concentrations and/or absorbance metrics) will be send to you | 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 :
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What type of sample would you like to analyze?
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Did you input a high or low amount of cells/tissue in the extraction?
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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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Are our standard deliverables ok?
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...
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 :
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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 a specific deliverable?
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Do we need to expedite (comes at extra cost)?
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...
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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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Follow our device dependent instructions : see details here
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Label your tubes/plates correctly : see details here
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Adhere to the corresponding transport conditions listed under Sample types
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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.
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When expedited, the turn-around-time (TAT) is 2 workdays
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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.
