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Ambion Top Ten
Top Ten Challenges for RNA Isolation
1. Isolating RNA for Microarray Analysis
RNA quality is the single most important factor in RNA amplification for microarray experiments. Intact RNA that contains trace contaminants is reverse transcribed poorly and yields less amplified RNA (aRNA) than pure samples. Conversely, RNA samples that are free of trace contaminants but are partially degraded [i.e., 28S:18S ribosomal RNA (rRNA) ratios from 1–1.8], yield high quality aRNA. So use a purification method that focuses on removal of contaminants—a combination of phenol-based and solid phase RNA extraction methods yields high quality RNA for successful array analysis. The RiboPure™ Kit combines a single reagent homogenization and organic extraction step with traditional glass fiber filter (GFF) purification to yield exceptionally pure RNA, free of proteins and lipids. The procedure is suited for processing samples that could clog filter cartridges, are difficult to disrupt, and/or have high levels of ribonuclease or other contaminants.
If you prefer to use messenger RNA (mRNA) to extend array sensitivity and minimize background, our Poly(A)Purist™ mRNA Isolation Kit features an optimized oligo(dT) hybridization that minimizes rRNA contamination, and yields highly purified mRNA ideal for cDNA probe synthesis.
2. Isolating RNA for RT-PCR
RNA isolation methods like acid phenol extraction, GFF purification, or single-step reagents all provide RNA of acceptable quality for RT-PCR. However, none of these methods has the ability to completely remove genomic DNA (gDNA) contamination from the RNA. DNA removal is critical as it can serve as a template during the PCR portion of the experiment, resulting in false positives and high background. This problem has been resolved by our RNAqueous®-4PCR Kit, which produces RNA that is completely free of gDNA contamination from samples as small as 1 mg or 100 cells. The RNAqueous technology involves binding total RNA to glass fiber filters in a microfuge tube format and includes reagents to remove DNA and the DNase used to destroy it.
3. Isolating RNA Containing Small RNAs
Most current RNA purification procedures rely on either organic extraction followed by alcohol precipitation, or adsorption of nucleic acid molecules on a GFF or other solid matrix. The former procedure requires alcohol precipitation which is time consuming and only inefficiently recovers small RNAs. The latter procedure results in the loss of substantial amounts of small RNA. The mirVana™ miRNA Isolation Kit and mirVana™ PARIS™ Kit (for RNA and protein isolation) employ organic extraction followed by GFF purification using specialized binding and wash solutions specifically developed to maximize small RNA recovery. The mirVana technology effectively recovers all RNA—from large mRNA and rRNA down to 10mers—from virtually all cells and tissues.
4. Isolating RNA for High Throughput
More laboratories than ever are engaged in high throughput applications where the efficient processing of large numbers of samples is a challenge. Many of these processes call for repetitive execution of standard procedures requiring precision and reproducibility. Thus protocols should be easily optimized for both manual and automated high throughput applications. The fastest and safest RNA isolation methods available are either filter- or magnetic bead-based. Due to ease of handling and reduced cross contamination risks, the following kits are ideal for working with multiple samples. The RNAqueous®-96 Automated Kit is designed for processing small samples, from 100–5x105 cells or 0.1–1 mg tissue using a GFF to provide high yields of intact RNA. An optional, on-filter DNase treatment ensures the removal of gDNA for subsequent RT-PCR. The MagMAX™-96 Total RNA Isolation Kit is optimized for high throughput isolation of high quality, intact total RNA from 25–2 x106 cells and from small plant and mammalian tissue samples. TURBO DNase™ enzyme is included for complete gDNA removal. This technology has been optimized for both manual and automated high throughput processing. The MagMAX-96 for Microarrays Total RNA Isolation Kit combines organic extraction with magnetic bead-based purification and is designed for rapid, high throughput isolation of total RNA from mammalian cells and tissues for array analysis. The technology is optimized for use manually, with multi-channel pipettors, as well as with robotic liquid handlers.
5. Isolating RNA from Blood Samples
Blood is the most commonly used tissue in clinical research, yet it presents several challenges for RNA isolation: 1) it is difficult to stabilize RNA in blood samples collected in the field, 2) the complex cellular nature of blood makes it difficult to detect differential gene expression that occurs in only a subset of cell types, and 3) blood includes contaminants that can compromise down stream applications. To avoid interference from enzymatic inhibitors found in whole blood (e.g., heme) and from anticoagulants used to make analysis of blood samples possible (e.g., heparin), RNA purification from whole blood requires a stringent method that provides high quality RNA that has been depleted of inhibiting agents and contaminants.
The Applied Biosystems Tempus™ Tubes are designed for direct lysis of 3 mL of blood in a plastic, evacuated blood collection tube, which allows the transcript profile to be stable in the tube for 5 days at room temperature. RNA can be quickly isolated by centrifugation or vacuum filtration methods. The LeukoLOCK™ Total RNA Isolation System allows cellular fractionation of whole blood and total RNA stabilization and extraction from the leukocyte population. Recovery is 10–20 µg of RNA/10 mL of whole blood. The RiboPure™-Blood Kit purifies RNA directly from whole blood using samples that are 500 µL or less. By combining phenol/chloroform extraction, GFF-based purification, and DNase treatment, the procedure removes protein, heme, gDNA, and RNases, resulting in high yields of pure RNA ideal for RT-PCR. The included RNAlater® Tissue Collection: RNA Stabilization Solution ensures the stabilization of RNA within the whole blood sample prior to processing.
6. Isolating RNA from FFPE Tissue Samples
The ability to isolate RNA from archived tissue samples that is suitable for molecular analysis enables retrospective studies of diseased tissue. While preservation techniques use formaldehyde to maintain tissue structure and prevent putrefaction, this makes it difficult to perform molecular analyses on the samples. RNA is trapped and modified through protein-protein and protein-nucleic acid crosslinks.
RNA is also fragmented and chemically modified in formaldehyde or paraformaldehyde-fixed, paraffin-embedded (FFPE) tissues. The degree of RNA fragmentation that has already occurred in FFPE tissues cannot be reversed. However, the protease digestion conditions of our RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues are designed to release RNA fragments of all sizes, including microRNA, in 3 hours. The FFPE samples are deparaffinized, subjected to rigorous protease digestion, purified using GFF methodology that includes an on-filter nuclease treatment. The recovered RNA is suitable for microarray analyses, real-time RT-PCR, and mutation screening.
7. Removing Genomic DNA Contamination
Genomic DNA contamination can lead to inaccurate RNA quantitation and affect downstream applications, e.g., provide false positive results in RT-PCR-based protocols. DNase treatment is the simplest solution to this problem. However, subsequent inactivation of DNase I by heating can damage the RNA, and DNase I removal by phenol extraction can result in RNA loss. Another method for reducing gDNA contamination involves lithium chloride (LiCl) precipitation. This techniques can be used to preferentially precipitate the RNA, and is sufficient for template preparation for transcription but will not remove gDNA sufficiently for RT-PCR.
As an alternative to these techniques, DNA-free™ DNase Treatment and Removal Reagents provides a way to both remove gDNA from RNA samples and subsequently remove DNase I and divalent cations from the reaction mixture. It uses a novel DNase Removal reagent requiring no organic extraction, EDTA addition, or heat inactivation.
8. Working with Impure RNA
Pure RNA is the best substrate for RT-PCR. However, due to the extreme sensitivity of RT-PCR, when an RNA sample does contain a contaminant that inhibits enzymatic reactions, the sample can often just be diluted, which likewise dilutes the contaminant so that it no longer compromises the reaction.
RNA for array analysis, on the other hand, should be free of contaminating proteins, DNA, and cellular material, as well as phenol, ethanol, and salts associated with RNA isolation. An A260:A280 ratio of 1.7–2.1 indicates pure RNA. This level of purity can be obtained using isolation protocols that bind the RNA sample to glass fibers or beads, allowing thorough removal of contaminating components present in the sample (see Tip 1 above).
9. Working with Low Quality RNA
The success of RT-PCR performed on degraded RNA depends on the PCR primers, amplicon size, probes used for detection, and the extent of the RNA degradation. RT-PCR amplicons are usually designed to be short (60–100 bp), making it possible for successful use even with partially degraded RNA. If possible, design primers and probes to more than one area on the target mRNA to increase the number of templates amplified.
10. Improving RNA Recovery
Coprecipitants are used for precipitating small amounts of nucleic acids. They can make nucleic acid recovery quantitative and are especially important when working with small amounts of critical RNA samples. It is crucial to ensure that the coprecipitants are nuclease-free to minimize the risk of introducing nucleases into samples. Coprecipitants should also not affect downstream analyses. Applied Biosystems, now including Ambion, offers several high quality coprecipitants including Glycogen, GlycoBlue™ coprecipitant (glycogen derivatized with a blue dye to increase pellet visibility), sheared Yeast Total RNA, Yeast tRNA, and Linear Acrylamide.
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