Reagent Kits

AATI's capillary electrophoresis instruments utilize specially designed reagent kits to analyze diverse nucleic acid samples, from small RNA to genomic DNA, and NGS library quality control.

Reagent Kit Box

High Sensitivity RNA Analysis Kit (15 nt)

High Sensitivity RNA Analysis Kit (15 nt)
500 Sample High Sensitivity RNA Analysis Kit (15 nt)

The High Sensitivity RNA Analysis Kit (15 nt) is used to assess the integrity of total RNA and messenger RNA (mRNA) samples. Efficient analysis of RNA is crucial to many applications, including next-generation sequencing technologies, microarray analysis, qPCR, and Northern blot analysis. To expedite post-electrophoresis analysis, PROSize® To further enhance the ease of sample analysis post-electrophoresis, PROSize calculates the RNA Quality Number, a quality metric that enables efficient assessment of sample suitability for downstream applications. With a quantitative measure of RNA quality and high-throughput capability, this kit is ideal for any RNA analysis application.

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Components

Components 500 Sample Kit 1,000 Sample Kit
RNA Separation Gel 240 mL (DNF-265-0240) 500 mL (DNF-265-0500)
Intercalating Dye 30 µL (DNF-600-U030) 30 µL x 2 (DNF-600-U030)
5X 930 dsDNA Inlet Buffer 125 mL (DNF-355-0125) 300 mL (DNF-355-0300)
5X Capillary Conditioning Solution 50 mL (DNF-475-0050) 100 mL (DNF-475-0100)
HS RNA Diluent Marker (15 nt) 4 mL x 3 (DNF-370-0004) 4 mL x 6 (DNF-370-0004)
HS RNA Ladder 15 µL (DNF-386-U015) 15 µL x 2 (DNF-386-U015)
0.25X TE Rinse Buffer 250 mL (DNF-497-0250) 250 mL (DNF-497-0250)
BF-1 Blank Solution 8 mL (DNF-301-0008) 8 mL (DNF-301-0008)
Eppendorf LoBind® 0.5 mL tubes package of 50 package of 50

Specifications

Specifications Description
Total RNA mRNA
Sample Volume Required 2 µL
Diluent Marker Volume Required 18 µL
Sizing Range 200 nt – 6,000 nt
Sizing Accuracy ± 20%
Sizing Precision 20% CV
Limit of Detection (S/N > 3) 50 pg/µL 250 pg/µL
Quatitative Range (per smear) 50 pg/µL - 5000 pg/µL 500 pg/µL - 5000 pg/µL
Quantification Accuracy ± 30%
Quantification Precision 20% CV
Total Electrophoresis Run Time 31 min (Ultra-Short Array, 22-47); 40 minutes (Short Array, 33-55); 70 minutes (Long Array, 55-80)

Examples

The High Sensitivity RNA Ladder is shown below. The ladder was run on a Fragment Analyzer equipped with a Short Capillary Array (33-55). Cleanly defined and separated peaks provide accurate quantification and sizing for RNA samples that fall into the high sensitivity detection range.

Figure 1
Figure 1. HS RNA Ladder (DNF-386) separated on a Fragment Analyzer equipped with a Short Capillary Array (33-55) using the High Sensitivity RNA Analysis Kit (DNF-472).

Post-separation sample analysis is simplified with the RNA Property Summary function. Each summary includes RNA concentration, ratio between 28S and 18S ribosomal RNA (rRNA), and the RNA Quality Number (RQN). PROSize allows the user to select the RNA source as Eukaryotic, Prokaryotic, or Plant, providing the most common rRNA subunit sizes for each category in the RNA Property Summary. The rRNA ratio provides a measure of purity of the sample, ideal values fall between 1.5 and 2.0 for this metric. The RQN is the definitive quantitative measurement of RNA quality. High quality RNA suitable for sensitive downstream applications, such as sequencing, has an RQN of 7 or higher. Figure 2 depicts a high quality total RNA sample extracted from a eukaryotic sample, while Figure 3 depicts low quality total RNA, extracted from yeast.

Figure 2
Figure 2. High quality total RNA extracted from yeast separated on a Fragment Analyzer equipped with a Short Capillary Array (33-55) using the High Sensitivity RNA Analysis Kit (DNF-472). The Small RNA Region (also known as the 5S region) is well defined, as are the small and large rRNA subunits, providing a high RQN of 9.5.
Figure 3
Figure 3. Poor quality total RNA extracted from yeast. The separation was performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55) using the High Sensitivity RNA Analysis Kit (DNF-472). Poor quality RNA is indicated by the electropherogram, which does not show the Small RNA Region (5S region) or the small and large rRNA subunits. The poor quality of the RNA sample is supported by the low RQN value of 2.0.

The evaluation of RNA quality following ribosome depletion is an important step when investigating the transcriptome using sophisticated downstream methods such as RNA-Seq or microarrays, among other techniques. The presence of ribosomal RNA (rRNA) in an mRNA sample has a negative impact on downstream applications. In the case of RNA-Seq the presence of rRNA reduces sequencing depth in turn hiding rare transcripts. As such, pure mRNA is required for transcriptomic studies. The High Sensitivity RNA Analysis Kit (DNF-472) quickly and accurately measures the percentage rRNA contamination, depicted in the mRNA Property Summary feature in PROSize, as shown in figure 4. If there is rRNA contamination, depletion should be repeated with subsequent analysis prior to use in downstream applications.

Figure 4
Figure 4. Separation of an rRNA depleted total RNA sample performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55) using the High Sensitivity RNA Analysis Kit (DNF-472) using the parameters for an mRNA separation. Post-separation PROSize calculates the level of rRNA contamination, labeled in the figure.

Accurate quantification is an important metric of nucleic acid analysis. The High Sensitivity RNA Analysis Kit (DNF-472) provides accurate quantification, measuring sample concentrations in line with a standard fluorescent method. As shown in Figure 5, the quantification accuracy of the Fragment Analyzer provides equivalent measurements to a standard fluorometric instrument.

Figure 5
Figure 5. Fluorometric Instrument concentration plotted against Fragment Analyzer concentration for the same samples. A tight linear relationship and high R2 support the equivalency of the measured concentration between the two instruments.
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