TITLE: Quant-iT™ RiboGreen™ RNA Quantification
AUTHORS: Roey Angel, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The following protocol is intended for the quantification of RNA using </span><a href="https://www.thermofisher.com/order/catalog/product/R11490#/R11490" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Quant-iT™ RiboGreen™ RNA Assay Kit</span></a><span> (ThermoFisher). This protocol is a simplified and condensed version of the </span><a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fmp11490.pdf&title=UXVhbnQtaVQgUmlib0dyZWVuIFJOQSBSZWFnZW50IGFuZCBLaXQ=" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">full protocol</span></a><span> from the manufacturer.</span></div><div class = "text-block">The procedure described here is for 96 reactions. If samples are run in duplicates, then this should allow quantifying 40 samples.</div></div>

[STEPS]
?. [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.
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.
Quant-iT™ RiboGreen® RNA reagent is light sensitive and should be protected from light at all times.
?. [Prepare the reaction]
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.
[20X TE buffer]
[nuclease-free water]
?. [Prepare the reaction]
For high-range quantification:Dilute the E.coli rRNA-standard stock solution (100 μg ml-1) to a final concentration of 2 ng μl-1 by mixing 10 μl of rRNA-standard stock solution with 490 μl 1X TE buffer.For low-range quantification:Prepare a 20-fold dilution of the 2 ng μl-1 rRNA-standard work solution by mixing 10 μl of the 2 ng μl-1 rRNA-standard work solution with 190 μl 1X TE buffer to yield 0.1 ng µl-1 rRNA-standard work solution.
[rRNA-standard stock solution]
[1X TE buffer]
[diluted rRNA-standard solution]
[1X TE buffer]
?. [Prepare the reaction]
If needed, prepare a dilution of each sample in 1X TE so that the reading will be within the dynamic range.
It is advisable to run samples in duplicates for a more accurate quantification
?. [Prepare the reaction]
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.
?. [Prepare the reaction]
Prepare one of the following standard mixtures in the first two columns of a black, sterile, 96-well plate:  ABCD1Assay versionDiluted RNA std. (µl)1X TE (µl)Final RNA amount (ng)2High-range (4-200 ng µl-1)10002003Use 2 ng μl-1 standard50501004109020529846010007Low-range (200 pg µl - 10 ng µl-1)1000108Use 0.1 ng μl-1 standard50505910901102980.21101000
ABCD1Assay versionDiluted RNA std. (µl)1X TE (µl)Final RNA amount (ng)2High-range (4-200 ng µl-1)10002003Use 2 ng μl-1 standard50501004109020529846010007Low-range (200 pg µl - 10 ng µl-1)1000108Use 0.1 ng μl-1 standard50505910901102980.21101000
?. [Prepare the reaction]
Pipette 99 µl of 1X TE buffer in the remaining wells.
[1X TE buffer ]
Tip: use a mechanical or electronic dispenser during this step and step no. 9 to speed up the work.
?. [Prepare the reaction]
Pipette 1 µl of the unknown RNA samples in the remaining wells.
[Unknown RNA sample]
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.
?. [Prepare the reaction]
Pipette 100 µl of the RiboGreen® work solution into each well, including the standard and unknown sample wells
[RicoGreen work solution]
?. [Prepare the reaction]
Protect the 96-well plate from light and incubate for 2-5 min at room temperature.
?. [Measure the samples]
Place the plate in a plate reader and measure the fluorescence according to the following parameters:Excitation                   ~480 nmEmission                    ~520 nmIntegration time            40 sLag time                         0 sGain                                OptimalNumber of flashes      10Calculated well             highest standardShaking                          5 s
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.
?. [Measure the samples]
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). Calculate 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 each sample was used.
Do not forget to account for any dilutions when calculating the concentration of the RNA in the unknown samples