Small Nucleic Acid Solutions on the 2100 Bioanalyzer
Small Nucleic Acid Solutions on the 2100 Bioanalyzer
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The new Small RNA Kit is designed to quantify and monitor total miRNA fraction contents
(~ 20 nt) in total RNA or enriched small RNA samples, along with resolving all other small RNA components. The assay provides PAGE-level resolution, with the speed and ease of use offered by the 2100 bioanalyzer. Use it in combination with the RNA 6000 Kits first to evaluate total RNA integrity and potential gDNA contamination, and then check the small RNA fraction in detail (size and concentration of the subcomponents in the 6-150 nt range) for intact or purified samples.
Agilent’s bioanalyzer RNA solutions provide quick answers with just 1 µl of sample. The RNA 6000 Kits and RIN are widely accepted as the most reliable tools for total RNA integrity measurement. Excellent sensitivity and sample compatibility have made them indispensable for the quality control (QC) of gene expression protocols, for both microarrays and qPCR. The Small RNA Kit combines exceptional resolution to analyze small RNA fractions, up to 150 nt, with the “pico” sensitivity required to analyze cellular components in very low-abundance. The detection limit for total miRNA is less than 100 pg.
Small RNA molecules have been proven to play a crucial regulatory role in cellular processes such as apoptosis, proliferation, and differentiation, yet the lack of adequate analytical methods has hindered accurate downstream analysis. Agilent’s Small RNA Kit, the RNA 6000 Pico Chip, and the 2100 bioanalyzer enable high sensitivity evaluation of total RNA and characterization of small RNA fractions through two complimentary assays (Gassman et al 2007; see poster link below). The new Small RNA Kit provides you with the means to easily resolve and quantify small RNAs (6 – 150 nt) from either intact or purified samples. Good quantification estimates are now possible for miRNA, tRNA, and other small RNA fractions, using as little as 1 ng of total RNA in a 1 µl sample. The Small RNA Kit has been meticulously optimized to deliver high-resolution analysis, superior sensitivity, flexible sample compatibility, and significant enrichment of small RNA fractions. The new assay runs on the Agilent 2100 bioanalyzer, and utilizes a novel sieving matrix formulation and a dye system for analysis of both single and double stranded nucleic acids.
Figure 1. High-resolution analysis is demonstrated by separation of the small RNA ladder.
The small RNA ladder is a mixture of synthetic non-repetitive RNA oligo sequences. Notice the consistent spacing of the ladder peaks, demonstrating consistent resolution throughout the size range. The assay has been optimized to achieve higher sensitivity in the microRNA (miRNA) region.
Figure 2. Two complementary assays for the characterization of RNA for miRNA expression experiments.
Total RNA analysis of thymus samples with the RNA 6000 assay (A) and small RNA fractions with the new kit (B), using the same sample for both assays. The blue box (Panel A) indicates the sample size range shown in Panel B. The RNA 6000 assay provides information on integrity (RIN and ribosomal ratios), purity, and total RNA concentration, while the small RNA assay focuses on the small RNA region, resolving its components and characterizing miRNAs. The dominant small RNA species are tRNA (approximately 75 nt), 5s rRNA (100-120 nt), and 5.8s rRNA (140-160 nt). miRNA (15-30 nt) and other additional minor peaks are visible (most are degradation products from the larger 18S/28S ribosomal peaks). It should be noted that the large ribosomal bands are effectively excluded from the analysis with the small RNA assay.
Figure 3. Enrichment of small RNA fractions can be measured to monitor purification processes.
The small RNA assay is an invaluable tool to follow purification or enrichment experiments. By monitoring the concentration of miRNA and other sample fractions, enrichment factors and recoveries can be calculated, enabling the comparison and subsequent optimization of sample preparation protocols. Figure 3 depicts the analysis of total RNA isolated from a J774 cell line using Trizol (glycogen was added as a carrier). The total RNA sample was analyzed (440 ng/µl) and then purified in 2 steps. First, all RNA fractions below 200 nt were enriched using a commercially available kit (Qiagen). The primary RNA component of this sample was tRNA. In a second step, a further enrichment of samples below 40 nt was carried out using a polyacrylamide gel electrophoresis system with a subsequent gel elution. Although a significant portion of tRNA was still visible in the sample, there was approximately a 20-30-fold increase in the enrichment of RNA species in the 10-40 nt size range as compared to the tRNA fraction.
For information on the Agilent Small RNA Kit, review a detailed slide set to learn more.
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