View this free article featured in BioProbes 77 – Assay RNA quality with the updated Qubit benchtop fluorometer or download full issue of BioProbes 77 (PDF, 2.5 MB).
Introducing the breakthrough Invitrogen Qubit RNA IQ Assay, developed to quickly assess the quality and integrity of an RNA sample. This assay allows assessment of RNA quality at a lower cost and with an easier, simpler, and faster workflow than other solutions currently on the market.
How to use the Qubit 4 Fluorometer and Qubit RNA IQ assay to measure RNA integrity and quality.
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.
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.
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 2. A proprietary algorithm is used to report a quality score representative of the ratio of small and large and/or structured RNA in the sample. The score is a value from 1 to 10, similar to other RNA quality scores. With the Qubit RNA IQ Assay, a small number indicates that the sample consists of mainly small RNA (A), and a larger number indicates that the sample consists of mainly large RNA or RNA with tertiary structure (B).
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.
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.
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.
|Instrument and assay||Qubit 4 Fluorometer and RNA IQ Assay||Agilent 2100 Bioanalyzer™
instrument and RNA Nano Chip*
|Equipment and reagents required for assay*||
|Equipment set up||Turn on Qubit 4 Fluorometer||
|Sample prep time||~5 minutes||~30-45 minutes|
|~1 hour||Use chip within 5 minutes|
|Analysis run time||<4 seconds per sample||30 minutes per chip|
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.|
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.
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|
For Research Use Only. Not for use in diagnostic procedures.