Rapid, accurate RNA quality and integrity assessment

The new Invitrogen Qubit RNA IQ Kit and Qubit 4 Fluorometer work together to accurately distinguish viable from degraded data in just two steps. No special handling, tedious sample preparation, or waiting for results—just add as little as 1 uL containing 0.5-1.5 ug/uL (or containing 500-1500 ng/uL) of your sample and get your results typically in <4 seconds per sample.

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Quality and integrity data from a simple, 2-dye assay

The Qubit RNA IQ Assay utilizes two unique dyes—one that binds to large, intact and/or structured RNA, the other selectively binds to small, degraded RNA (Figure 1). Together they are able to quickly assess the quality and integrity of an RNA sample. To use, simply add your samples to the RNA IQ working solution, then measure on the Qubit 4 Fluorometer. Results are presented as a total value for the RNA sample integrity and quality or RNA IQ# and as the calculated % of large and small RNA in the sample (Figure 2). The RNA IQ# is based on a scale of 1 to 10 similar to other RNA quality scores, wherein a high IQ# indicates the majority of the sample consists of large and/or structured RNA. Conversely a small IQ# indicates the sample comprises mainly small RNA with limited tertiary structure (Figure 3).


Figure 1. Selectivity of the RNA IQ reagents for large and small RNA. Triplicate samples containing 100 ng/mL rRNA (E. coli) and varying amounts of siRNA (0 to 50 ng/μL) were assayed with the Qubit RNA IQ assay (Q33221, Q33222) on the Qubit 4 Fluorometer. Relative fluorescent units (RFUs) (A) and IQ#’s were plotted (B) for these samples.  
 

Qubit 4 Fluorometer and Qubit RNA IQ Assay results

Figure 2. Qubit 4 Fluorometer and Qubit RNA IQ Assay results.
 

Figure 3 Qubit RNA IQ fluorescence response and IQ# for solutions containing various amounts of large and small RNA. Triplicate samples containing a total of 100 ng/mL RNA, comprising small RNA (Silencer Select GAPDH siRNA) and large RNA (rRNA, E. coli) were assayed with the Qubit RNA IQ assay (Cat. Nos. Q33221, Q33222) on the Qubit 4 Fluorometer. Relative fluorescent units (RFUs) (A) and IQ#s were plotted (B) for these samples.

The Qubit 4 Fluorometer and RNA IQ Assay go together

Our newest Qubit Fluorometer—the Qubit 4 Fluorometer—was engineered specifically to accommodate the RNA IQ Assay in addition to all previous Qubit quantitation assays. It’s important to note that the RNA IQ assay cannot be performed on the original Qubit, Qubit 2.0 or Qubit 3.0 Fluorometers. 

Invitrogen Qubit 4 and RNA Quantitation and Qualification

Comparing RNA IQ solution–based method to electrophoresis methods

The Qubit RNA IQ provides a fast, simple method to check whether a RNA sample has degraded (Figure 4). A gel or microfluidic-based electrophoresis is still recommended to obtain detailed fragment size information or size distribution of the RNA sample. RNA IQ assay results reflect the percentage of large and/or structured and small RNA in the sample, plus correlate well with electrophoretic methods (Figure 5), however it is important to note that the IQ#s reflect the percentage of large and small RNA in the sample, thus there will be some differences between the IQ# and other quality scores dependent upon how the other quality scores are calculated (Figure 6). Initially recommend running RNA samples with the RNA IQ assay and the traditional electrophoretic method to ascertain how the measurements relate to specific samples or downstream applications. The primary difference between the solution-based RNA IQ assay to traditional electrophoresis-based techniques is the amount of time it takes to ascertain whether an RNA sample has degraded. Gels are considered easy to perform, but still take time to run. Microfluidic-based methods not only take time to obtain results, but often have lengthy procedures to prepare the samples (Table 1). 

Figure 4.  Real-time RNA degradation monitored with Qubit RNA IQ Assay.  Triplicate samples of 100 ng/μL rRNA solutions were incubated with RNase A at the specified time points RNase OUT was added to inhibit RNase activity. Samples were measured with the Qubit RNA IQ Assay (Cat. Nos. Q33221, Q33222) on the Qubit 4 Fluorometer. The graphs on the right show the RFU values with low amounts of RNase (top right), where the large and/or structured dye fluorescence maintains its signal initially as the RNA is degraded, coupled with increasing fluorescence from the small RNA dye. The bottom graph shows that at higher concentrations of RNase, the large and/or structured dye fluorescence decreases much more rapidly, with of course increasing fluorescence from the small RNA dye. 

Figure 5. Assessment of rRNA integrity and quality following treatment with RNase measured with either Qubit RNA IQ Assay or Agilent Bioanalyzer™ instrument. 750 fM Rnase A was added to aliquots of a 100 ng/mL solution of rRNA (E. coli), and at various time points  treated with RNase OUT then measured with either the Qubit RNA IQ Assay with a Qubit 4 Fluorometer (A) or Agilent Bioanalyzer™ Prokaryote Total RNA Nano chip (B, C, D).

Figure 6.  Comparison of RNA integrity and quality using Qubit RNA IQ assay and Agilent Bioanalyzer™ instrument with total RNA measured after 1 minute treatment with RNase.  rRNA was treated with 0.5pg/µL RNase A, followed by the addition of RNaseOUT after 1 minute.  Samples were measured either on a Prokaryote Total RNA Nano Chip or with the Qubit RNA IQ Assay (Cat. Nos. Q33221, Q33222) on the Qubit 4 Fluorometer. The difference between the quality scores, IQ#: 9.8 and RIN#: 8.9 is the result of signal from 5s rRNA and tRNA that exhibit tertiary structure and also bind the large RNA dye.
 

Table 1. Comparing the Qubit 4 Fluorometer method with Agilent Bioanalzyer for measuring RNA sample quality. 

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
  • Qubit RNA IQ assay kit
  •  Pipettor
  • Qubit Assay tubes
  • Agilent 2100 Bioanalyzer instrument
  • RNA 6000 Nano Assay Kit
  • Pipettor
  • Microcentrifuge tubes
  • Chip priming station
  • IKA vortexer
  • Microcentrifuge
  • Computer
  • Heating block or water bath
  • RNase-free water
Equipment set up 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
  2. Add sample [1-20 uL (0.5-1.5 ug)] and standards to Qubit RNA IQ working solution
  1. Prepare RNA ladder
  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.

Complete RNA sample intelligence with NanoDrop and Qubit combination

The reliability and reproducibility of any RNA analysis requires starting with RNA of known quantity, quality and purity. The combination of the Qubit RNA IQ assay on the Qubit 4 Fluorometer together with a UV-absorbance measurement using a NanoDrop Spectrophotometer provides one of the fastest and easiest methods to obtain quantity, quality and purity of an RNA sample. The NanoDrop Spectrophotometer requires just 1 µL of sample and by simply measuring at 230, 260 and 280 nm, you can obtain the total amount of nucleic acids present (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) and HS (high-sensitivity) assays can also be used for RNA quantitation. These assays include dyes that selectively bind only to intact RNA, thus can be used for pure RNA samples or samples containing DNA that also absorb at 260 nm. 


Qubit 4 Fluorometer guide

Choose the Qubit 4 Fluorometer configuration that suits your assay needs.

Qubit 4 Fluorometer Qubit 4 Quantitation Starter Kit Qubit 4 NGS Starter Kit Qubit 4 RNA IQ Starter Kit
  • Qubit 4 Fluorometer
  • Qubit 4 Fluorometer User Guide
  • Qubit 4 Fluorometer Quick Reference Card
  • Universal power cord with 4 adaptor plugs
  • Qubit 4 USB cable
  • Qubit 4 USB flash drive
  • Qubit screen cleaning cloth
  • One Qubit 4 Fluorometer
  • Qubit assay tubes (set of 500)
  • Qubit dsDNA BR Assay Kit, 100 assays
  • Qubit dsDNA HS Assay Kit, 100 assays
  • Qubit RNA HS Assay Kit, 100 assays
  • Qubit RNA BR Assay Kit, 100 assays
  • Qubit RNA IQ Assay Kit, 75 assays
  • Qubit Protein Assay Kit, 100 assays
  • One Qubit 4 Fluorometer
  • Qubit assay tubes (set of 500)
  • Qubit dsDNA HS Assay Kit, 500 assays 
  • One Qubit 4 Fluorometer
  • Qubit assay tubes (set of 500)
  • Qubit RNA IQ Assay Kit, 275 assays
Q33226 Q33227 Q33228 Q33229

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