Analysis of small RNAs with the Agilent 2100 bioanalyzer

The Total RNA 6000 Nano Kit was used with the Agilent 2100 bioanalyzer to determine the integrity and the concentration of small RNA (i.e., 5.8S, 5S RNAs and tRNA) samples extracted with different protocols from various cell lines. The small RNAs fraction (<200 bp) should also contain microRNAs in their primitive (pri-miRNA), precursor (premiRNA) and mature (miRNA) forms. The data analysis software automatically reports the corresponding RNA concentrations for each sample in a range between 5 and 500 ng/μl. Moreover, the performance of Agilent 2100 bioanalyzer was compared to the most commonly used techniques for RNA separation, detection and quantitation. Comparisons between different techniques were based on sensitivity and quantitative accuracy. The advantages of detection sensitivity and accuracy, coupled with a rapid and automated system, indicate that analyses performed by Agilent 2100 bioanalyzer are superior to the leading alternatives. Application Note Andrea Masotti Viviana Caputo Sabrina Prudente Gian Franco Bottazzo lead to the loss of substantial amounts of miRNAs and other small RNAs. For example, if RNA is isolated using the traditional glass fiber filtration, it most likely lacks a complete representation of small RNAs. Therefore, it is of crucial importance to develop an efficient and accurate method to determine the integrity and the concentration of total and small RNAs, prepared with different RNA isolation procedures or when purchasing commercially available purified RNAs. The performance of the Agilent 2100 bioanalyzer to separate, detect and quantitate small RNAs is compared to the most commonly used techniques. Samples extracted with different protocols and from various cell lines were evaluated. Comparisons between techniques were based on sensitivity and quantitative accuracy. Materials and methods Total RNA extration and small RNAs enrichment protocols Total RNA (sample A) was extracted with MirVanaTM miRNA Isolation Kit (Ambion) according to manufacturer’s instructions. This kit also provides specific procedures to isolate and enrich small RNAs (LMW) (samples 2 and 4) from higher molecular weight (HMW) RNAs (i.e, 28S, 18S and 5.8S) (samples 1 and 3). Cell lines Two cell lines, one lymphoblastoid and one human brain tumor (U118), were cultured using standard procedures. Cells were 2 trypsinized and pelleted by centrifugation. Approximately 106 cells for each extraction were resuspended in the appropriate lysis solution contained in the kit and treated according to manufacturer’s instructions. Denaturing gels All samples (500 ng) were resolved into 15 % polyacrylamide TBE-Urea gel and 1 % agarose MOPS-formaldehyde gel stained with ethidium bromide. Agilent chip preparation RNA samples were analyzed with the Total RNA 6000 Nano Kit, specifically optimized for the analysis of total RNA with the Agilent 2100 bioanalyzer. The chips were loaded and run on the Agilent 2100 bioanalyzer according to manufacturer’s instructions . Agilent 2100 bioanalyzer instrument and software The Agilent 2100 expert B.02.02 software includes data collection, presentation and interpretation functions. Data can be displayed as a gel-like image and/or as electropherogram(s). The RNA profile is automatically displayed as individual electropherograms. An additional data evaluation tool is available for data comparison that allows to compare files from multiple chips. The Agilent 2100 bioanalyzer is equipped with independent high voltage power supplies connected to 16 platinum electrodes, which allow multiple and precisely controlled injections and separations. The instrument uses fluorescence detection, by monitoring the emission between 670 nm and 700 nm. Introduction Isolation of pure and intact RNA is essential for several applications but the concentration and integrity of total RNA preparations is significantly affected by the specific purification process used. Applications may include RT-PCR, preparations of targets for microarrays, ribonuclease protection assays, preparations of cDNA libraries and Northern blotting among others. Recently, the importance of low molecular weight RNAs (miRNAs, siRNAs, and snRNAs, etc.) has been outlined in different studies1. In particular, the importance of a new class of small RNA, namely microRNAs (miRNAs), has been highlighted. MicroRNAs are small RNA molecules encoded by the genomes of plants and animals. These highly conserved, ~21-mer RNAs regulate the expression of genes binding the 3'-untranslated regions (3'-UTR) of specific mRNAs. Each miRNA is thought to regulate multiple genes, and since hundreds of miRNA genes are predicted to be present in higher eukaryotes the potential regulatory circuitry afforded by them is enormous. It has been hypothesised that in higher eukaryotes, the role of miRNAs in regulating gene expression could be as important as the transcription factors. Therefore, techniques that allow an accurate detection of small RNA quality and quantity are highly important. Standard protocols for the isolation of total RNA and mRNA actually are not optimized for recovery of small RNA molecules and may Results and discussion We purified RNA from different samples, using two different RNA extraction procedures according to MirVanaTM miRNA Isolation Kit instructions. The first is designed to recover total RNA (sample A) and the other one, specific for small RNA enrichment, is able to efficiently separate RNA molecules of approximately 200 nt from the larger ones (samples 1 and 3). Following the latter procedure the resulting RNA preparations (samples 2 and 4) are highly enriched for low molecular weight (LMW) RNAs (5.8S, 5S, tRNA, miRNAs, siRNAs, and/or snRNAs both singleand double-stranded species). This enrichment protocol can be useful in all the experiments requiring the removal of predominant large RNAs, for example, for accurate analysis of miRNA expression by QPCR or microarray analysis. Moreover, an accurate quantitation is important for a lot of procedures including the determination of the correct amount of RNA template needed for RT-PCR and microarray labeling reactions or the minimum amount needed in a Northern blot. All RNA samples, obtained following these two protocols, were separated on a denaturing agarose and polyacrilamide gels. Briefly, each sample (500 ng) were loaded on 1 % agarose gel and stained with ethidium bromide in order to estimate the quality and quantity of RNA sample. Results were compared to the Agilent 2100 Bioanalyzer digital gel (figure 1A) and to the corresponding electropherograms. (figure 1B). Comparing the agarose gel sample electrophoreFigure 1 A) Agarose gel (1 %, left) of total RNA (sample A), small RNAs (LMW) (samples 2 and 4) and higher molecular weight (HMW) RNAs (samples 1 and 3) (500 ng per sample) extracted with MirVanaTM miRNA Isolation Kit (Ambion) compared to the digital gel (right) obtained with Agilent 2100 bioanalyzer (L=ladder) (500 ng per sample). B) Comparison of Agilent 2100 bioanalyzer electropherograms of sample A (total RNA), 1 (HMW RNA) and 2 (LMW RNA). Peak “a” (insert) was detected only in total and HMW RNA samples (A and 1) but not in LMW RNA (2) enriched sample. 3 Samples: 1 2 A 3 4 L 1 2 A 3 4