Apr 30, 2020
Version 2
Quant-iT™ RiboGreen™ RNA Quantification V.2
- 1Soil and Water Research Inrastructure
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: eva.petrova@bc.cas.cz
Protocol Citation: Roey Angel, Eva Petrova 2020. Quant-iT™ RiboGreen™ RNA Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.bfbvjin6
Manuscript citation:
Angel, R., and Conrad, R. (2013). Elucidating the microbial resuscitation cascade in biological soil crusts following a simulated rain event. Environ Microbiol 15, 2799–2815. doi:10.1111/1462-2920.12140.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 20, 2020
Last Modified: April 30, 2020
Protocol Integer ID: 35925
Abstract
The following protocol is intended for the quantification of RNA using Quant-iT™ RiboGreen™ RNA Assay Kit (ThermoFisher). This protocol is a simplified and condensed version of the full protocol from the manufacturer.
The procedure described here is for 96 reactions. If samples are run in duplicates, then this should allow quantifying 40 samples.
Attachments
mp11490.pdf
122KB
Materials
MATERIALS
Quant-iT™ RiboGreen™ RNA Assay KitInvitrogen - Thermo FisherCatalog #R11490
STEP MATERIALS
Quant-iT™ RiboGreen® RNA Assay KitThermo ScientificCatalog #R11490
Protocol materials
Quant-iT™ RiboGreen™ RNA Assay KitInvitrogen - Thermo FisherCatalog #R11490
Quant-iT™ RiboGreen® RNA Assay KitThermo ScientificCatalog #R11490
Quant-iT™ RiboGreen® RNA Assay KitThermo ScientificCatalog #R11490
Safety warnings
Quant-iT™ RiboGreen® RNA reagent is classified as Not Hazardous. Nevertheless, the user should always consult the MSDS accompanying any of the reagents and apparatus described in this protocol.
Before start
- This protocol is optimised for measuring an entire 96-well plate. It assumes that 10 wells will be used for measuring the standards and the blank samples (in duplicates) and 86 wells will be used for measuring unknown RNA samples (typically in duplicates).
- The protocol can be easily adjusted for a lower number of samples by reducing the volume of the working solutions of the reagents. Note though that enough TE should be retained for diluting the standard stock solution (490 or 680 µl), for potentially diluting the unknown samples, if their concentration is too high, and for accounting for pipetting errors. To fill the plate, 19.2 ml of TE is needed. So if only 40 wells are to be used for measuring unknown samples prepare about ml of TE buffer.
- The dynamic range of the assay is between 1 ng ml-1 to 50 ng ml-1 in the "low-range" version of the assay and 20 ng ml-1 to 1 µg ml-1 in the "high-range" version of the assay. This translates into RNA sample concentrations of 0.2-10 ng µl-1 and 4-200 ng µl-1 in the low-range and high-range assays, respectively. Samples with higher RNA concentration need to be diluted (e.g. in RNase-free water or TE buffer).
- Note that some compounds that often contaminate RNA are known to interfere with the measurement and produce a lower observed measurement. Please refer to the full protocol for a list of these compounds and their effect on the measurement.
- Quant-iT™ RiboGreen® reagent also binds to DNA. Samples containing DNA should be pre-treated with an RNase-free DNase before using this protocol to ensure that the fluorescent signal is solely due to binding with RNA.
Prepare the reaction
Prepare the reaction
Take out all reagents from the fridge and bring them to room temperature.
Take out the RNA samples from the freezer. RNA samples should be slowly thawed on ice.
Note
Quant-iT™ RiboGreen® RNA reagent is dissolved in dimethylsulfoxide (DMSO), which freezes below 19 °C. The reagent must be completely thawed before using it by bringing it to room temperature. After the reagent thawed, it is advisable to briefly vortex the tube to make sure it is adequately mixed and to spin it down in a centrifuge or a mini centrifuge.
Note
Quant-iT™ RiboGreen® RNA reagent is light sensitive and should be protected from light at all times.
Quant-iT™ RiboGreen® RNA Assay KitThermo ScientificCatalog #R11490
20m
Prepare 22 ml 1X TE buffer by pipetting 1.1 ml of 20X TE buffer into 20.9 ml of nuclease-free water into a sterile and nuclease-free 50 ml tube.
Mix by inverting the tube several times.
1.1 mL 20x TE buffer
20.9 mL nuclease-free water
2m
For high-range quantification:
Dilute the E.coli rRNA standard (100 μg ml-1) to a final concentration of 2 ng μl-1 by mixing 10 μl of rRNA standard–stock with 490 μl 1X TE.
For low-range quantification:
Prepare a 100 ng ml-1 standard by mixing 10 μl of the 2 ng μl-1 work rRNA standard solution with 190 μl 1X TE.
2m
If needed, prepare a dilution of each sample in 1X TE so that the reading will be within the dynamic range.
Note
It is advisable to run samples in duplicates for a more accurate quantification
Prepare the RiboGreen® work solution:
For the high-range assay pipette 50 μl Quant-iT™ RiboGreen® RNA Reagent and 9950 µl of 1X TE into a sterile and nucleic-acids free 50 ml tube.
For the low-range assay pipette 5 μl Quant-iT™ RiboGreen® RNA Reagent and 9995 µl of 1X TE into a sterile and nucleic-acids free 50 ml tube.
Mix by inverting and keep the solution away from light.
2m
Prepare one of the following standard mixtures in the first two columns of a black, sterile, 96-well plate:
| Assay version | Diluted RNA std. (µl) | 1X TE (µl) | Final RNA amount (ng) | |
| High-range (4-200 ng µl-1) | 100 | 0 | 200 | |
| Use 2 ng μl-1 standard | 50 | 50 | 100 | |
| 10 | 90 | 20 | ||
| 2 | 98 | 4 | ||
| 0 | 0 | 0 | ||
| Low-range (200 pg µl - 10 ng µl) | 100 | 0 | 10 | |
| Use 0.1 ng μl-1 standard | 50 | 50 | 5 | |
| 10 | 90 | 1 | ||
| 2 | 98 | 0.2 | ||
| 0 | 100 | 0 |
Equipment
96-Well microtiter plate
NAME
Polystyrene cell-culture plate
TYPE
Nunc
BRAND
165305
SKU
LINK
Black, flat-bottom, sterile
SPECIFICATIONS
10m
Pipette 99 µl of TE buffer in the remaining wells.
99 µL 1X TE buffer
Note
Tip: use a mechanical or electronic dispenser during this step and step no. 9 to speed up the work.
Equipment
Multipette® E3
NAME
Electronic dispenser
TYPE
eppendorf
BRAND
4987000010
SKU
LINK
2m
For low-concentration samples5 steps
To measure samples with a low concentration, it is possible to reduce the volume of the work solution at this step and pipette more sample in the next step, for a total volume of 100 µl.
Pipette 1 µl of the unknown RNA samples in the remaining wells.
1 µL Unknown RNA sample
Note
Use either a diluted sample in case the concentration is expected to be higher than the dynamic range limit or larger volume in case the concentration is expected to be below the detection limit.
5m
Pipette 100 µl of RiboGreen® work solution into each well, including the standard and unknown sample wells
100 µL RicoGreen work solution
2m
Protect the 96-well plate from light and incubate for 2-5 min at room temperature.
00:05:00
5m
Measure the samples
Measure the samples
5m
5m
Place the plate in a plate reader and measure the fluorescence according to the following parameters:
Excitation ~480 nm
Emission ~520 nm
Integration time 40 s
Lag time 0 s
Gain Optimal
Number of flashes 10
Calculated well highest standard
Shaking 5 s
Equipment
Infinite M Nano
NAME
Absorbance plate reader
TYPE
Tecan
BRAND
TEC006436I
SKU
LINK
Note
It is also possible to set the gain to a fixed value (e.g. 100). If the fluorescence values of the standard drop over time this could indicate damage to the reagents or the RNA standard.
5m
Plot the measured fluorescent values of the standard samples against their known concentrations and fit a linear curve using linear regression. Make sure that the coefficient of determination (R2) is close to 1 (typically >0.98). Calculte the RNA concentrations in the unknown samples using the slope and intercept parameters of the linear equation. Output values you obtained are in ng µl-1, assuming 1 µl of sample was used.
Note
Do not forget to account for any dilutions when calculating the concentration of the RNA in the unknown samples
10m