USER GUIDE

Quant-iT™ RiboGreen™ RNA Reagent and Kit
Catalog Numbers R11490, R11491, T11493
Pub. No. MAN0002073 Rev. A.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

Product description™

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The Quant-iT RiboGreen RNA Reagent is an ultrasensitive fluorescent nucleic acid stain for quantitating RNA in solution. Detectingand quantitating small amounts of RNA is important in many applications including measuring yields of in vitro transcribed RNA andmeasuring RNA concentrations before performing Northern blot analysis, S1 nuclease assays, RNase protection assays, cDNA library
preparation, reverse transcription PCR, and differential display PCR.

Figure 1 Dynamic range and sensitivity of the Quant-iT™ RiboGreen™ RNA Assay.
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For the high-range assay (top panel), the Quant-iT RiboGreen RNA Reagent was diluted 200‑fold into 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE)and 100 µL of the reagent solution was added to microplate wells containing 100 µL of ribosomal RNA in TE. For the low-range assay (bottom™
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panel), the Quant-iT RiboGreen RNA Reagent was diluted 2,000‑fold into TE and 100 µL of the reagent solution was added to microplate wells
containing 100 µL of ribosomal RNA in TE. Samples were excited at 485 ± 10 nm and the fluorescence emission intensity was measured at 530 ±12.5 nm using a fluorescence microplate reader. Fluorescence emission intensity was then plotted versus RNA concentration.
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The Quant-iT RiboGreen RNA Reagent enables quantitation of as little as 1 ng/mL RNA (200 pg RNA in a 200 µL assay volume) with a™
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fluorescence microplate reader using fluorescein excitation and emission wavelengths. The linear range of the Quant-iT RiboGreen RNA

For Research Use Only. Not for use in diagnostic procedures.

Reagent extends over three orders of magnitude in RNA concentration (1 ng/mL to 1 µg/mL) using two dye concentrations (Figure 1). The
high-range assay allows quantitation of 20 ng/mL to 1 µg/mL RNA, and the low-range assay allows quantitation of 1 ng/mL to 50 ng/mL
RNA. This linearity is maintained in the presence of several compounds commonly found to contaminate nucleic acid preparations,™
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including nucleotides, salts, urea, ethanol, chloroform, detergents, proteins, and agarose. Although the Quant-iT RiboGreen RNA
Reagent also binds to DNA, pretreatment of mixed samples with DNase can be used to generate an RNA-selective assay (see “Eliminate
DNA from samples” on page 5).

Contents and storage
Component

Quant-iT™ RiboGreen™
RNA Reagent[1]Quant-iT™ RiboGreen™
RNA Assay Kit

Cat. No. R11491

Cat. No. R11490

1 mL

1 mL

Quant-iT™ RiboGreen™ RNA
Reagent (Component A)

Concentration

Storage[2]Solution in DMSO

2°C to 8°C[3]DesiccateProtect from light

20X TE Buffer, RNase-free(Component B)

Not applicable

25 mL

200 mM Tris-HCl, 20 mM
EDTA, pH 7.5 in DEPCtreated water≤30°C

Ribosomal RNA standard,
16S and 23S rRNA from
E. coli (Component C)

Not applicable

5 × 200 µL

100 µg/mL in TE buffer

2°C to 8°C[4]Avoid freeze-thaw cycles

Number of labelings: 2,000 to 20,000 with an assay volume of 200 µL in a 96-well microplate format. The Quant-iT™ RiboGreen™ RNA Assay can beadapted for use in cuvettes or 384-well microplates.
Approximate fluorescence excitation/emission maxima: 500/525 nm, bound to nucleic acid.
[1]

Stand-alone reagent does not include Components B and C.
When stored as directed, products are stable for at least 6 months.
[3] For long-term storage, the Quant-iT™ RiboGreen™ RNA Reagent can be stored at ≤–20°C[4] For long-term storage, store the rRNA standards at ≤–20°C or –70°C.

[2]

Required materials not supplied• Nuclease-free pipettors and tips
• Nuclease-free water• Microplates for Fluorescence-based Assays, 96-well (Cat. No. M33089)

Prepare the assay buffer
Prepare the 1X TE working solution by diluting the concentrated buffer (Component B) 20‑fold with nuclease-free water. Prepare nucleasefree water by treating distilled, deionized water with 0.1% diethyl pyrocarbonate (DEPC), incubating for several hours at 37°C, andautoclaving for at least 15 minutes at 15 lbs/sq. inch to sterilize the water and eliminate DEPC.

IMPORTANT! TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is used to prepare the Quant-iT™ RiboGreen™ RNA Reagent and for

diluting RNA standards and samples. Make sure the TE solution is free of contaminating nucleases and nucleic acids. The 20X TE buffer™
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included in the Quant-iT RiboGreen RNA Assay Kit is nuclease-free and nucleic acid–free.

Prepare the reagent™

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Two different dye concentrations are required to achieve the full linear dynamic range of the Quant-iT RiboGreen RNA Assay. Before™
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preparing the working solution of the Quant-iT RiboGreen RNA Reagent, decide whether you wish to perform the high-range assay(20 ng/mL to 1 µg/mL RNA), low-range assay (1 ng/mL to 50 ng/mL RNA), or both.
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On the day of the experiment, allow the Quant-iT RiboGreen RNA Reagent to warm to room temperature before opening the vial,™
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then prepare an aqueous working solution of the Quant-iT RiboGreen RNA Reagent by diluting the concentrated DMSO stock solution(Component A) into TE, 200‑fold for the high-range assay or 2,000‑fold for the low-range assay. For microplate assays of a total 200 µL™
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assay volume, you need 100 µL of the Quant-iT RiboGreen RNA Reagent working solution per sample.
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For example, to prepare enough working solution to assay 100 samples in 200 µL volumes, add 50 µL Quant-iT RiboGreen RNA™
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Reagent to 9.95 mL TE for the high-range assay or add 5 µL Quant-iT RiboGreen RNA Reagent to 9.995 mL TE for the low-range
assay.
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Note: Allow the Quant-iT RiboGreen RNA Reagent to warm to room temperature before opening the vial. Cold DMSO solutions absorb
moisture from warmer, room temperature air, resulting in loss of efficacy for the reagent. Always store the DMSO stock solution in the2™

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Quant-iT RiboGreen RNA Reagent and Kit User Guide

presence of desiccant when not in use. We recommend preparing the working solution in sterile, disposable polypropylene plasticware™
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rather than glassware, as the reagent may adsorb to glass surfaces. Protect the working solution from light, as the Quant-iT RiboGreenRNA Reagent is susceptible to photodegradation. For best results, use the working solution within a few hours of preparation.

Prepare the RNA standard curve
1. Prepare a 2 µg/mL solution of RNA in TE using nuclease-free plasticware. Determine the RNA concentration on the basis ofabsorbance at 260 nm (A260) in a cuvette with a 1 cm pathlength; an A260 of 0.05 corresponds to 2 µg/mL RNA.
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The ribosomal RNA standard (Component C), provided at 100 µg/mL in the Quant-iT RiboGreen RNA Assay Kit, is diluted 50‑foldin TE to make the 2 µg/mL working solution. For example, 4 µL of the RNA standard mixed with 196 µL of TE is sufficient for thestandard curve described in step 2.
Note: For a standard curve, we commonly use 16S and 23S ribosomal RNA, although any purified RNA preparation may be used. Itis sometimes preferable to prepare the standard curve with RNA similar to the type being assayed. However, most single-stranded
RNA molecules yield approximately equivalent signals.
Note: The RNA solution used to prepare the standard curve should be treated the same way as the experimental samples andshould contain similar levels of contaminants. See “Effects of common contaminants” on page 4 for a list of contaminants tested™
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in the Quant-iT RiboGreen assay.
2. For the high-range standard curve, dilute the 2 µg/mL RNA solution into microplate wells as shown in Table 1. For the low-range
standard curve, dilute the 2 µg/mL RNA solution 20‑fold into TE to make a 100 ng/mL RNA stock solution, then prepare the dilution
series shown in Table 2.
Table 1 Protocol for preparing a high-range standard curve.
Volume of TE buffer

Volume of 2 µg/mL RNA stock

Volume of 200‑fold diluted
Quant-iT™ RiboGreen™ RNAReagent

Final RNA concentration in theassay

0 µL

100 µL100 µL

1 µg/mL

50 µL

50 µL

100 µL500 ng/mL

90 µL

10 µL

100 µL

100 ng/mL

98 µL

2 µL

100 µL

20 ng/mL

100 µL0 µL

100 µL

blank

Table 2 Protocol for preparing a low-range standard curve.
Volume of TE buffer

Volume of 100 ng/mL RNA
stock

Volume of 2,000‑fold diluted
Quant-iT™ RiboGreen™ RNA
Reagent

Final RNA concentration in the
assay

0 µL

100 µL100 µL

50 ng/mL

50 µL

50 µL

100 µL

25 ng/mL

90 µL

10 µL

100 µL

5 ng/mL

98 µL

2 µL

100 µL

1 ng/mL

100 µL0 µL

100 µLblank™

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3. Add 100 µL of the appropriate aqueous working solution of Quant-iT RiboGreen RNA Reagent (prepared in “Prepare the reagent”
on page 2) to each microplate well. Use the high-range working solution for performing the high-range assay, and use the low-range
working solution for performing the low-range assay. Mix well and incubate for 2–5 minutes at room temperature, protected fromlight.
4. Measure the sample fluorescence using a fluorescence microplate reader and standard fluorescein wavelengths (excitation∼480 nm, emission ∼520 nm).
Note: To ensure that the sample readings remain in the detection range, set the instrument's gain so that the sample containing thehighest RNA concentration yields a fluorescence intensity near the microplate reader’s maximum. For optimal detection sensitivity,
the instrument gain can be increased for the low-range assay relative to the high-range assay. To minimize photobleaching effects,
keep the time for fluorescence measurement constant for all samples.
5. Subtract the fluorescence value of the reagent blank from that of each of the samples. Use corrected data to generate a standard
curve of fluorescence versus RNA concentration (see Figure 1).

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Quant-iT RiboGreen RNA Reagent and Kit User Guide

3

Analyze samples
1. Dilute the experimental RNA solution in TE to a final volume of 100 μL in microplate wells.
Note: You can alter the amount of sample diluted, provided that the final volume remains 100 μL. High dilutions of the experimental
sample may serve to diminish the interfering effect of certain contaminants. However, extremely small sample volumes should beavoided because they are difficult to pipet accurately. In addition, the level of assay contaminants should be kept as uniform as
possible throughout an experiment, to minimize sample-to-sample signal variation. For example, if a series of RNA samples containwidely differing salt concentrations, then they cannot be compared to a single standard curve. To avoid this problem, simply adjust
the concentration of contaminants to be the same in all samples, if possible (see “Effects of common contaminants” on page 4).
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2. Add 100 μL of the aqueous working solution of the Quant-iT RiboGreen RNA Reagent (prepared in “Prepare the reagent” onpage 2) to each sample. Incubate for 2–5 minutes at room temperature, protected from light.
3. Measure the fluorescence of the samples using the same instrument parameters used to generate the standard curve (see step 4).
To minimize photobleaching effects, keep the time for fluorescence measurement constant for all samples.
4. Subtract the fluorescence value of the reagent blank from that of each of the samples. Determine the RNA concentration of thesample from the standard curve generated in “Prepare the RNA standard curve” on page 3.
5. The assay can be repeated using a different dilution of the sample to confirm the quantitation results.

Effects of common contaminants™

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The Quant-iT RiboGreen Assay remains linear in the presence of several compounds that commonly contaminate nucleic acidpreparations, although the signal intensity may be affected (Table 3). For the highest accuracy, the standards should be prepared under
the same conditions as the experimental samples and contain similar levels of contaminants.
Table 3 Effects of common contaminants on the signal intensity of the assay.
Maximum acceptable concentration% Signal change[1]Ammonium acetate

20 mM

4% decrease

Sodium acetate

20 mM

11% decrease

Sodium chloride

20 mM

15% decrease

Zinc chloride

1 mM

9% decrease

Magnesium chloride

0.5 mM

9% decrease

Calcium chloride

0.1 mM

2% increase

Cesium chloride

10 mM

8% decrease

Guanidinium thiocyanate

10 mM

9% decrease

Urea

3M

13% decrease

Phenol

0.5%

5% decrease

Ethanol

20%

10% decrease

Chloroform2%

15% increase

Sodium dodecyl sulfate

0.05%

10% decrease

Triton™ X-100

0.5%

8% decrease

Bovine serum albumin

0.2%

11% decrease

IgG

0.02%

4% decrease

CompoundSalts

Organic solvents

DetergentsProteins4™

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Quant-iT RiboGreen RNA Reagent and Kit User Guide

Maximum acceptable concentration% Signal change[1]Formamide

10%

12% decrease

Sucrose>500 mM

4% decrease

Boric acid

100 mM

15% decrease

10%10% decrease

CompoundOther compounds

Polyethylene glycol
Agarose[1]

0.2%

3% increase

The compounds were incubated at the indicated concentrations with the Quant-iT RiboGreen RNA Reagent in the presence of 1.0 mg/mL ribosomal RNA. All samples wereassayed in a final volume of 200 µL in 96‑well microplates using a fluorescence microplate reader. Samples were excited at 485 ± 10 nm and fluorescence intensity was measured
at 530 ± 12.5 nm.
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Eliminate DNA from samples
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The Quant-iT RiboGreen RNA Reagent also binds to DNA. Fluorescence in samples that is due to the Quant-iT RiboGreenRNA Reagent binding to DNA can be eliminated by pre-treating the sample with RNase-free DNase, ensuring that the entire sample
fluorescence is due to dye bound to RNA.
1. Prepare 10X DNase digestion buffer: nuclease-free 200 mM Tris-HCl, pH 7.5, containing 100 mM MgCl2 and 20 mM CaCl2.
2. Add 0.11 volume of 10X DNase digestion buffer to each DNA-containing sample (for example, to a 9 µL sample, add 1 µL 10Xbuffer).
3. Add ∼5 units of RNase-free DNase I per µg of DNA estimated to be in the sample.
4. Incubate the sample at 37°C for 90 minutes.
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5. Dilute the sample at least 10‑fold into TE to diminish effects of the digestion buffer salts on the Quant-iT RiboGreen assay
procedure.
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6. Perform the Quant-iT RiboGreen assay as described (see “Analyze samples” on page 4).

Related products
Table 4 Bulk Reagents and Kits
QuantityCat. No.

1 mL assay kit

P7589

10 x 100 µL

P11496

1 mL reagent

P7581

10 x 100 µL

P11495

100 mLT11493

Quant-iT™ RiboGreen™ RNA Assay Kit

1 mL assay kit

R11490™

Quant-iT RiboGreen RNA Reagent

1 mL reagent

R11491

Quant-iT™ RediPlate™ 96 RiboGreen™ RNA
Quantitation Kit

1 plateR32700Quant-iT™ OliGreen™ ssDNA Assay Kit

1 mL assay kit

O11492

Quant-iT™ OliGreen™ ssDNA Assay Reagent

1 mL reagent

O7582

ProductQuant-iT™ PicoGreen™ dsDNA Assay Kit

Quant-iT PicoGreen dsDNA Reagent™

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TE Buffer (20X), RNase-free

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Quant-iT RiboGreen RNA Reagent and Kit User Guide5

Table 5 Microplate Reader Assays
Product

Dynamic Range

QuantityCat. No.

Quant-iT 1X dsDNA Assay Kit,
High Sensitivity

200 pg−100 ng

1,000 reactions

Q33232

Quant-iT™ 1X dsDNA Assay Kit,
Broad-Range

4 ng−2 μg

1,000 reactions

Q33267Quant-iT™ DNA Assay Kit, HighSensitivity

200 pg−100 ng

1,000 reactions

Q33120Quant-iT™ DNA Assay Kit, BroadRange

4 ng−1 μg

1,000 reactions

Q33130Quant-iT™ RNA Assay Kit

5−100 ng

1,000 reactions

Q33140Quant-iT RNA Reagent

5−100 ng

1,000 reactions

Q32884

Quant-iT™ RNA Assay Kit, BroadRange

20 ng−1 μg

1,000 reactions

Q10213

Quant-iT™ RNA XR Assay Kit

200 ng−10 μg

1,000 reactions

Q33225

Quant-iT™ microRNA Assay Kit

1−100 ng

1,000 reactions

Q32882

Quant-iT™ Protein Assay Kit

250 ng−5 μg

1,000 reactions

Q33210—10 plates

M33089™

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Microplates for Fluorescence-based
Assays, 96-wellLimited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms andConditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, pleasecontact Life Technologies at www.thermofisher.com/support.
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The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0002073
Revision

Date

A.0

15 March 2022

1.00

10 June 2008

Description
The format and content were updated. The version numbering was reset to A.0 inconformance with internal document control.
New document for the Quant-iT™ RiboGreen™ RNA Assay Kit and Quant-iT™ RiboGreen™
RNA Reagent.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
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