Quickly assess RNA integrity and quality

The Qubit RNA IQ Assay was developed to quickly assess the integrity and quality (IQ) of an RNA sample. It uses two dyes: one that binds to large, intact, and/or structured RNA, the other to small, degraded RNA.

RNA quality assay

Product

No. of assays

Cat. No.

Qubit RNA IQ Assay Kit

75

Q33221

275

Q33222

Qubit RNA IQ Standards

1 set

Q33235

Simple protocol

To use the Qubit RNA IQ Assay, simply add as little as 1 µL of your samples (containing 0.5–1.5 µg RNA) to the RNA IQ working solution and measure on the Qubit 4 or Qubit Flex Fluorometer. No special handling, tedious sample preparation, or waiting for results is required. A sample’s IQ score, a number between 1 and 10, represents the percentage of large to small RNA molecules in the sample.

Qubit RNA IQ assay results

 

The RNA IQ score is a value from 1 to 10, similar to other RNA quality scores. Based on a proprietary algorithm, it represents the ratio of large and/or structured RNA to small RNA in the sample. A low score (A) indicates that the sample consists of mainly small RNA while a high score (B) indicates that the sample consists mainly of large and/or structured RNA.

As shown in the table, the Qubit RNA IQ Assay can be far simpler and faster to use than other methods such as the Agilent™ Bioanalyzer™ system. In addition, the Qubit RNA IQ score is more consistent with RT-qPCR measures of RNA quality than the Bioanalyzer RNA integrity number (RNA), as demonstrated on the Sample Data page.

Protocols for measuring RNA sample quality with the Qubit RNA IQ Assay and the Agilent Bioanalyzer

Instrument and assay

Qubit 4 Fluorometer and RNA IQ Assay

 Agilent 2100 Bioanalyzer™ instrument and RNA Nano Chip*

Equipment and reagents required for assay*

Qubit 4 Fluorometer

Agilent 2100 Bioanalyzer instrument

Qubit RNA IQ assay kit

RNA 6000 Nano Assay Kit

 Pipettor

Pipettor

Qubit Assay tubes

Microcentrifuge tubes

 

Chip priming station

 

IKA vortexer

 

Microcentrifuge

 

Computer

 

Heating block or water bath

 

RNase-free water

Equipment setup

Turn on Qubit 4 Fluorometer

Replace the syringe at the chip priming station with each new DNA kit

Adjust the base plate of the chip priming station

Adjust the syringe clip at the chip priming station

Adjust the Bioanalyzer chip selector

Set up the vortex mixer

Start the software before you load the chip

Sample prep time

~5 minutes

~30-45 minutes

Sample prep

1. Prepare Qubit RNA IQ working solution by adding RNA IQ dye to RNA IQ buffer

1. Prepare RNA ladder

2. Add sample [1-20 uL (0.5­–1.5 ug)] and standards to Qubit RNA IQ working solution

2. Prepare the gel (step includes 10 min centrifugation)

 

3. Prepare the gel-dye mix (step includes 10 min. centrifugation)

 

4. Load the gel-dye mix into the chip

 

5. Load marker into the chip

 

6. Load the RNA ladder and sample(s) (step includes 1 min IKA vortexer) 

Sample stability

~1 hour 

Use chip within 5 minutes

Analysis run time

<4 seconds per sample

30 minutes per chip

Regular maintenance

None

Check the performance of the chip priming station by applying the seal test on a monthly basis

For electrophoresis assays: clean electrodes on a daily basis using the electrode cleaner

Thoroughly clean electrodes on a monthly basis using a toothbrush and distilled water

Clean the focusing lens once a month (or after any liquid spill) using isopropanol

Product(s) required for maintenance

None 

RNaseZAP (electrode cleaning)

* Compiled from Agilent G2938-90037 and G2946-90003 user manuals and Thermo Fisher Scientific MAN0017210 quick reference card.

Sample data

This data example shows that the two reagents in the Qubit RNA IQ Assay are highly specific to large and small RNA respectively, and that RNA IQ scores increase appropriately as the percentages of large RNA increase.

Selectivity of the RNA IQ assay reagents for large and small RNA

Triplicate samples containing 100 ng/mL rRNA (E. coli) and varying amounts of siRNA (0–50 ng/µL) were assayed with the Qubit RNA IQ assay on the Qubit 4 Fluorometer. Graphs show (A) relative fluorescence units (RFUs) and (B) IQ scores for these samples. The data shows that the dyes are specific to large (like rRNA) and small (like siRNA) RNA respectively, and that IQ scores are strongly correlated with the percentage of large RNA in the sample.

This data example shows that, with exposure to RNase A, an RNA sample degrades over time and its RNA IQ score drops. The degradation is faster with higher doses of RNase A, as would be expected.

rRNA degradation by RNase A over time, measured by the Qubit RNA IQ Assay

Triplicate samples of 100 ng/mL rRNA solutions were incubated with different doses of RNase A in the final assay solution containing multiplexed dyes and assay buffer. (A) Higher doses of RNase A caused faster degradation of RNA over 60 minutes as reflected by its RNA IQ score. (B) Exposure to 10 fM RNase A caused a gradual decline in large RNA fluorescence over the hour and a corresponding increase in small RNA fluorescence. (C) With exposure to 100 fM RNase A, the changes occurred more quickly.

Combine Qubit fluorometry with NanoDrop UV fluorescence to measure RNA quantity, quality, and purity

The reliability and reproducibility of any RNA analysis requires starting with RNA of known quantity, quality, and purity. Combining the Qubit RNA IQ assay on the Qubit 4 or Flex Fluorometer and a UV absorbance measurement using a NanoDrop Spectrophotometer provides one of the fastest and easiest methods to assess the quantity, quality, and purity of an RNA sample.

NanoDrop Spectrophotometers require just 1 µL of sample. With three simple measurements, you can obtain the total amount of nucleic acids (260 nm), any protein contamination (280 nm), and any phenol or other solvent contaminants (230 nm) present in the sample.

The Qubit RNA XR (extended range), BR (broad range), and HS (high sensitivity) assays can be used for RNA quantitation. These assays include dyes that selectively bind only to intact RNA and thus can be used for pure RNA samples as well as samples containing DNA that also absorb at 260 nm.

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