DNA AND RNA QUANTIFICATION

ABOUT

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 :

When having ultra low DNA inputs, PCR-free library preparations are off the table
Ultra high DNA (or RNA) inputs may lead to inhibition of certain enzymatic steps (e.g. fragmentation / cDNA synthesis)
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
...

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.

DNA/RNA qualification

METHODOLOGY

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 :

A260/280 : indicative for protein, phenol or other aromatic compound contamination
- Normal range DNA : 1,7 - 1,8
- Normal range RNA : 1,8 - 2,0
A260/230 : indicative for EDTA, carbohydrate, chaotropic salts (and also protein) contamination
- Normal range DNA and RNA : >1,8 (preferentially >2,0)

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

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

Specifcations

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

Wavelengths

Run time

Sensitivity

Invitrogen Qubit 2.0

DNA

RNA

Qubit dsDNA BR
Qubit dsDNA HS
Quantifluor ONE

Qubit RNA BR

2-1000 ng/µl*
0,05-60 ng/µl*
0,4-200 ng/µl*

5-600 ng/µl*

Excitation :
430-495 nm
600-645 nm
Emission :
510-580 nm
665-720 nm

ca. 2s / sample

Link

Perkin Elmer DropletQuant

DNA/RNA

Lunatic Plate

1,5-2000 ng/µl

230-750 nm

ca. 5 min / 96 samples (plate)

Link

Perkin Elmer Victor Nivo 3F

DNA

Qubit dsDNA BR

0,2-400 ng/µl**

Installed filters :
315-395 nm
395-415 nm
430-490 nm
450-510 nm
500-560 nm
700 nm
740-760 nm

ca. 4 min / 96 samples (plate)

Link

* 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

CERTIFICATES

Part of accredited process

DOWNLOADS

Downloads

CONTACTS

Contacts

BRIGHTcore general inquires

info@brightcore.be

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

UZ Brussel

BRIGHTcore

Laarbeeklaan 101

1090 Brussels

SAMPLE TYPES

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.

Information on storage

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

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

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?
Did you input a high or low amount of cells/tissue in the extraction?
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

Start here

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

Get Quote

Submit your samples

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

Fill out our sample submission form
1. Send it via email : contact us
2. Print out the form and include it with your samples
Send your samples to us
1. Follow our device dependent instructions : see details here
2. Label your tubes/plates correctly : see details here
3. Adhere to the corresponding transport conditions listed under Sample types
Indicate if we can discard your samples after completion of project

3

Submission form

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.