Document Sample
RNA Kit Powered By Docstoc
					RNA Purification
There is a saying in many labs: “Garbage in, garbage out”. Since what goes “in” to so many assays, arrays, and reactions is RNA, you want to be sure that you are using RNA of the highest quality in order to ensure quality results downstream. Whether you need to isolate mRNA, microRNA or total RNA, make sure to choose a kit that is appropriate for your sample type. Some kits work across a range of sample types, while others have been developed for specific needs, such as extraction from FFPE tissue. You may also want to consider how much handling a kit requires (i.e. how many steps in the protocol) as well as whether organic solvents are required. Fortunately, there are a myriad of kits available for the extraction of high quality RNA. You are sure to find one that suits your particular needs. You can start your search with the links below and the related products to the right. Source -

RNA Isolation Basics
Obtaining high quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments, including Northern analysis, nuclease protection assays, RT-PCR, RNA mapping, in vitro translation and cDNA library construction. To be successful, however, the RNA isolation procedure should include some important steps both before and after the actual RNA purification. The following article discusses various RNA isolation procedures and ways of increasing RNA yields. Tissue or Cell Sample Collection and Disruption Our ongoing research into optimizing RNA analysis has identified two points in the RNA isolation process that can be improved; treatment and handling of tissue or cells prior to RNA isolation and storage of the isolated RNA. Since most of the actual RNA isolation procedure takes place in a strong denaturant (e.g. GITC, LiCl, SDS, phenol) that renders RNases inactive, it is typically prior to, and after the isolation, when RNA integrity is at risk. Finding the most appropriate method of cell or tissue disruption for your specific starting material is important for maximizing the yield and quality of your RNA preparation. See the article "Cell Disruption - Getting the RNA Out," which describes various disruption methods and suggests which method to use for specific tissues / cell types, for more information on this subject. During tissue disruption for RNA isolation, it is crucial that the denaturant be in contact with the cellular contents at the moment that the cells are disrupted. This can be problematic when tissues/cells are hard (e.g. bone, roots), when they contain capsules or walls (e.g. yeast, Gram-positive bacteria) or, when samples are numerous, making rapid processing difficult. A common solution to these problems is to freeze the tissue/cells in liquid nitrogen or on dry ice. The samples must then be ground with a mortar and pestle into a fine powder, which is added to the denaturant. While this freezing and grinding process allows the researcher to postpone RNA isolation, it is a time consuming and laborious process. Ambion now offers a completely new type of product, a tissue storage solution that provides more flexibility and time. RNAlater® Tissue Storage:RNA Stabilization Solution allows the researcher to postpone RNA isolation for days, weeks, or even months after tissue collection without sacrificing the integrity of the RNA. Dissected tissue or collected cells are simply dropped into the RNAlater solution at room temperature. The solution permeates the cells,

stabilizing the RNA. The samples are then stored at 4°C. Samples can be shipped on wet ice or even at room temperature if shipped overnight! Figure 1 shows the integrity of RNA isolated from tissues stored in RNAlater at 4°C, room temperature and even at 37°C. Samples stored at 4°C generate intact RNA even after storage for a month.

Source -

Options for RNA Isolation Ambion's family of RNA isolation kits provide flexibility for sample size and type, and include kits for the isolation of total or poly(A) RNA. The accompanying table — "Which RNA Isolation Kit to Choose?" — summarizes the advantages of each kit to help you determine the optimal RNA Isolation Kit for your particular application. This table also gives information on typical yield per reaction. For additional information on approximately how much total or poly(A) RNA can be recovered from a given amount of tissue or cells, as well as rough expression levels for rare to moderately abundant transcripts, see "Tissue/Cells to RNA Conversions." TRI Reagent® is a single, homogenous solution for the isolation of total RNA. This phenolbased reagent contains a unique combination of denaturants and RNase inhibitors and is used in a convenient, single-step disruption/separation procedure. The tissue or cell sample is homogenized or disrupted in the TRI Reagent, chloroform is mixed with the lysate, and the mixture is separated into three phases by centrifugation. The RNA is then precipitated from the aqueous phase with isopropanol. The entire procedure can be completed in no more than one hour to produce high yields of intact RNA for use in Northerns, nuclease protection assays, RT-PCR and in vitro translation. TRI Reagent is especially effective for purifying RNA from microorganisms. Ambion's RNAqueous® Technology is a rapid, filter-based RNA isolation system that does not require the use of phenol, chloroform or other toxic organic chemicals. The entire procedure can be completed in 20 to 30 minutes, depending on the time required for tissue disruption (see an example of typical results in Figure 4). RNAqueous Technology-based Kits are available in both small and large scale formats. The RNAqueous Kit is designed for sample sizes of 10 to 75 mg of tissue or 106 to 107 cells, whereas the RNAqueous-Midi Kit is designed for tissue samples of up to 0.5 g or 109 cells. The RNAqueous-96 Kit utilizes a 96well plate format for high-throughput RNA isolation from 100 to 2 x 106 cells or 0.1 to 1.5 mg of tissue. The RNAqueous-4PCR Kit provides RNA free of genomic DNA contamination from samples as small as 1 mg or 100 cells. Ambion's Plant RNA Isolation Aid is recommended for use with the RNAqueous Kit for purification of total RNA from plant tissues. Alternatively, total RNA may be isolated using Ambion's ToTALLY RNA™ Kit. This procedure is similar to the widely used guanidinium thiocyanate/acid phenol:chloroform method but has been modified to include two formulations of phenol:chloroform and an

optional LiCl precipitation step. The modifications help to reduce or eliminate DNA, carbohydrate, heme and other contaminants that can otherwise be difficult to separate from the RNA. Although more time consuming than other RNA isolation procedures — it can take up to 85 minutes from tissue/cell disruption to RNA — reactions can be scaled up or down to accommodate large or small samples. The Paraffin Block RNA Isolation Kit allows easy isolation of RNA from formalin-fixed, paraffin-embedded tissue sections for use in RT-PCR. The fast, 4-hour protocol yields RTPCR competent RNA even for rare messages (Figure 5). Paraffin-embedded tissue blocks as old as 16 years have yielded amplifiable RNA. Source -

Prokaryotic Total RNA
Speed is critical in the purification of bacterial RNA due to the short half-life of bacterial mRNA and the need to rapidly "freeze" the mRNA expression profile. Some bacterial isolation protocols call for the pretreatment of bacteria with lytic enzymes (which are usually used in conjunction with a one-step isolation reagent such as TRI Reagent). While this pretreatment does assist lysis, it delays isolation and may lead to altered expression profiles. The following methods are better alternatives for effectively freezing gene expression profiles:
 

immediate cell lysis and RNA purification rapid freezing in liquid nitrogen (a freeze-thaw treatment may help with lysis of some bacteria) resuspension of cells in Ambion's RNAlater


Easily lysed Gram-negative bacteria may be pipetted directly into a boiling lysis buffer of choice (without even removing the culture medium), and RNA can be immediately extracted with TRI Reagent. Most other bacteria will need to be pelleted by brief centrifugation prior to the above treatments. To purify RNA from a bacterial cell pellet (including frozen pellets and those stored in RNAlater), add boiling lysis buffer to the pellet and vortex rapidly, then immediately extract the lysate with TRI Reagent or hot acid phenol:chloroform. Harsh mechanical devices (e.g. bead mills) may be required to disrupt some bacterial species. Once lysed, extract the preparation with hot acid phenol:chloroform or TRI Reagent. Alternatively, it may be possible to disrupt bacteria directly in TRI Reagent or acid phenol:chloroform using a bead mill. For more information see "Purify Bacterial RNA". mRNA Eukaryotic mRNA Poly(A) RNA (mRNA) makes up between 1-5% of total cellular RNA and is most frequently used for 1) detection and quantitation of extremely rare mRNAs, 2) synthesis of probes for array analysis, and 3) the construction of random-primed cDNA libraries, where the use of total RNA would generate rRNA templates that would significantly dilute out cDNAs of interest. Removal of ribosomal and transfer RNA results in up to a 30-fold enrichment of a

specific message. Figure 6 demonstrates the enrichment of a specific message seen after selecting for poly(A) RNA. Source -

RNA Kits
Ambion's Poly(A)Purist™ Kits make isolation of mRNA easy by providing a rapid method for isolating the highest possible purity mRNA from total RNA, with the highest possible yield. The Poly(A)Purist and MicroPoly(A)Purist Kits include premeasured aliquots of oligo(dT) cellulose. The Poly(A)Purist MAG Kit utilizes oligo(dT) magnetic bead-based purification. These kits use an optimized hybridization protocol so that mRNA is efficiently bound but without co-isolation of rRNA. These procedures usually require only a single round of oligo(dT) selection to yield mRNA for even the most stringent applications. Prokaryotic mRNA For decades mRNA has been isolated from eukaryotic sources using oligo(dT) selection. Bacteria, however, lack the relatively stable poly(A) tails found on eukaryotic messages. Until very recently, isolating mRNA from bacteria has been virtually impossible. The MICROBExpress™ Bacterial mRNA Isolation Kit employs a novel technology to remove >95% of the 16S and 23S rRNA from total RNA of E. coli and other bacterial species The kit is suitable for rapid mRNA purification from a broad spectrum of Gram-positive and Gram-negative bacteria. mRNA isolated with MICROBExpress is a superior template for synthesizing labeled cDNA for array analysis (Figure 7) and is ideal for quantitative RTPCR, Northern blotting, and cDNA library construction. In the first step of the MICROBExpress procedure, bacterial total RNA is mixed with an optimized set of capture oligonucleotides that bind to the bacterial 16S and 23S rRNAs. Next, the rRNA is removed from the solution using derivatized magnetic microbeads. The mRNA remains in the supernatant and is recovered by ethanol precipitation. The entire procedure takes less than 2 hours. Source -

Storage of Isolated RNA
The last step in every RNA isolation protocol, whether for total or mRNA preparation, is to resuspend the purified RNA pellet. After painstakingly preparing an RNA sample, it is crucial that RNA be suspended and stored in a safe, RNase-free solution. Ambion now has several RNA Storage Solutions for this purpose:
 

THE RNA Storage Solution (1 mM sodium citrate, pH 6.4 ± 0.2) 0.1 mM EDTA (in DEPC-treated ultrapure water)

 

TE Buffer (10mM Tris-HCl, 1 mM EDTA, pH 7.0) RNAsecure™ Resuspension Solution

Our technical service staff has received numerous requests for pre-made 0.1 mM EDTA and TE Buffers; these solutions are often specified in common RNA isolation and analysis protocols. These storage solutions are ideal for researchers who already use them but would like the convenience and security of having them premade and certified RNase-free. We are also introducing THE RNA Storage Solution, a buffer which delivers greater RNA stability than 0.1 mM EDTA or TE. THE RNA Storage Solution has two features which minimize base hydrolysis of RNA: a low pH, and sodium citrate, which acts both as pH buffer and a chelating agent (divalent cations catalyze base hydrolysis of RNA). THE RNA Storage Solution is compatible with all of the common RNA applications such as reverse transcription, in vitro translation, Northern analysis, and nuclease protection assays. The RNAsecure Reagent is a unique nonenzymatic reagent for the irreversible inactivation of RNases in enzymatic reactions. RNAsecureª Resuspension Solution contains the same active ingredients as the RNAsecure Reagent, but is supplied at a working concentration for direct resuspension of RNA pellets. To inactivate RNases, the RNA pellet is resuspended in the RNAsecure Resuspension Solution and heated to 60°C for 10 minutes. A unique feature of RNAsecure is that reheating after the initial treatment will reactivate the RNase-destroying agent to eliminate any new contaminants. Source -

Total RNA Isolation Mini Kit
Isolate RNA in a straightforward way The Agilent Total RNA Isolation Mini Kit uses a straightforward, spin-column method to deliver total RNA for use in gene expression and other downstream analyses. A unique prefiltration column simultaneously clarifies and homogenizes viscous samples, facilitating simultaneous RNA isolation and genomic DNA removal. The kit delivers significant reduction of genomic DNA contamination compared to silica-based kits and it's easier to use and simpler than conventional DNAse-based methods.  Sufficient reagents for 50 isolations
   

Sample required per isolation is 2.5 - 30 mg tissue or 5 x 105 - 1 x 106 cells Process time is approximately 20 minutes Delivers intact and high-purity total RNA in as little as 10 µL Delivers up to a 1,000-fold reduction of genomic DNA contamination without DNase treatment Handles a wide range of sample types (e.g., mammalian cells and tissues including fibrous samples, yeast, and bacteria).



An optional, on-column DNase digestion step is available that easily integrates into the workflow Contents include columns, tubes, and key reagents Kit should be stored at room temperature

 

Source -

RNA isolation kit
ARCTURUS BIOSCIENCE Arcturus Bioscience PicoPure RNA Isolation Kit was used in RNA isolation in order to investigate the regulatory role of TGF-β on several VIC functions that occur during the early stages of wound repair. to the paper

ARCTURUS Arcturus PicoPure RNA isolation kit was used in RNA isolation in order to identify a tumorigenic subpopulation within the p53 null mammary tumors. to the paper Arcturus PicoPure RNA Isolation Kit was used in RNA isolation in order to study the role of meprinβ in the form of glomerulonephritis. to the paper

ARCTURUS/MOLECULAR DEVICES Arcturus/Molecular PicoPure RNA Isolation Kit was used in RNA isolation in order to demonstrate that Sca-1 localizes to lipid microdomains in myogenic precursors and that it associates with a catalytically active intracellular messenger IDE. to the paper

stratagene search stratagene RNA isolation kit products Stratagene RNA isolation kit was used to prepare mRNA in order to study the effect of the role for Anaplasma phagocytophilum in increasing cathepsin L activity on neutrophil function Source -

Isolation of High-Quality Total RNA from a Broad Range of Sample Material
Introduction The MagNA Pure Compact Instrument together with the new MagNA Pure Compact RNA Isolation Kit can be used for automated isolation of total RNA from blood, blood cells, tissue, and cultured cells. The MagNA Pure Compact System perfectly fits into Roche Applied Science's integrated solution for real-time PCR: The MagNA Lyser Instrument, Universal ProbeLibrary, Transcriptor First Strand cDNA Synthesis Kit and FastStart TaqMan® Probe Master. The basic procedure of the RNA isolation is based on the MagNA Pure Magnetic Glass Particle (MGP) Technology. The principle steps include lysis of samples in a special tissue lysis buffer containing chaotropic salt and proteinase K that destroys remaining proteins including nucleases. Nucleic acids are immobilized on the MGP surfaces and genomic DNA is degraded by incubation with DNase. Unbound substances are removed by several washing steps and finally, purified RNA is eluted from the MGPs. The aim of this work was to assess the efficacy of the MagNA Pure Compact RNA Isolation Kit to extract total RNA from different tissues and cell lines. In addition to the assessment of purity and integrity, the suitability of extracted RNA to down-stream applications such as real-time PCR and microarray-based gene expression analysis was tested.

Materials and Methods Tissue and cell samples SK-N-DZ cells (human neuroblastoma; ATCC no. CRL-2149) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 4 mM lglutamine. PC 12 cells (rat pheochromocytoma; ATCC no. CRL-1721) were cultured in Ham’s F12K medium supplemented with 15% horse serum, 2.5% fetal bovine serum and 2 mM l-glutamine. Human tissue samples were obtained from the Department of Pathology at the University Hospital of North Norway, Tromsø. Animal tissue samples were obtained from Sprague-Dawley rats. Animals were maintained in our animal facilities on standard laboratory chow. Isolation of RNA Tissue specimens were either used directly, fresh-frozen, or stored in RNA Later Solution. Disruption and homogenization of tissue samples were performed using the MagNA Lyser Instrument, according to the instructions. Cell culture samples were directly homogenized in PBS and lysis buffer included in the kit. The elution volume was set to 50 µl.

Quantity and purity of total RNA RNA was quantified measuring absorbance at 260 nm and RNA purity was determined by the ratios OD260 nm/280 nm and OD260 nm/230 nm using the NanoDrop instrument. The RNA integrity (RIN) was determined by electrophoresis using the Agilent Bioanalyzer 2100. Quantification of mRNA by real-time RT-PCR of cyclophilin A

Three microliters of the eluted RNA were reversely transcribed with Transcriptor First Strand cDNA Synthesis Kit. The real-time PCR assay targeting cyclophilin A was performed with 250 ng cDNA using a real-time PCR instrument, FastStart TaqMan® Probe Master, and Universal ProbeLibrary probes (human probe #48 and rat probe #42). As a negative control, RNA was replaced by water. To prove the absence of residual genomic DNA within RNA extractions, RNA samples were directly subjected to PCR targeting cyclophilin A using 2 ng of human genomic DNA as positive control. Microarray experiments In three independent dye-flip experiments, 3 µg of total RNA from rat liver and rat lung were labeled with Cy3 and/or Cy5 using the 3 DNA Array 350 HS Kit. Hybridizations were performed with a TECAN 4000HS instrument. Rat oligo arrays were purchased from the Norwegian Microarray Consortium (NMC). Experiments were performed at the Microarray Resource Centre Tromsø (MRCT). The arrays were normalized using three dye-flip replicates. This reduced the set of six arrays into three normalized versions. M-M (log-ratio versus log-ratio) scatter plots of the dye-normalized replicates were then produced. The correlation coefficients of the replicates were finally calculated using the Pearson correlation function. .

Results and Applications To measure the quality and amount of total RNA extracted using the MagNA Pure Compact Instrument and the MagNA Pure Compact RNA Isolation Kit, we measured the absorbance at 230 nm, 260 nm and 280 nm, conducted LabChip (Agilent) microfluidic analysis and performed quantitative real-time PCR of cyclophilin A. In addition, we tested the suitability of the extracted total RNA for microarray analysis. We demonstrated that total RNA preparations from different human and rat tissues and from cell lines isolated with the MagNa Pure Compact Instrument yield excellent RNA in sufficient amounts. Electrophoresis and the ratios of OD260 nm/280 nm show that high quality total RNA was extracted (Figure 1, Table 1). The integrity (RIN) of RNA samples was in a very good range with respect to the different tissues tested. The lower OD260 nm/230 nm ratio of isolations from rat brain is most likely due to the relatively high lipid and fatty acid composition in brain tissue. The reproducibility of RNA extraction was evaluated by real-time PCR for RNA extracted from human colon. The low CVs of crossing points calculated by quantification analysis of the amplification curves showed good reproducibility of the isolation procedure (Figure 2a). All preparations were virtually free of residual genomic DNA (Figure 2b) and no signs of PCR inhibition were observed (Figure 2a). In addition, dye-flip microarray experiments with RNA from rat liver versus rat lung displayed high log-ratio correlations between replicates, indicating that the extracted RNA is suitable for differential expression studies (Figure 3). The correlation coefficients were calculated using all available features on the arrays. Therefore, the values indicate a high degree of consistency between replicates Conclusions In conclusion, the MagNA Pure Compact Instrument and MagNA Pure Compact RNA Isolation Kit facilitate the preparation of highly purified total RNA from fresh and fresh-

frozen animal tissue as well as cultured cell samples. Tissue samples stabilized by specific reagents (e.g., RNA Later) can also be used. The total time for automated purification of RNA from eight samples is approximately 35 minutes. The purified RNA from tissues and cells is an ideal starting material for down-stream applications such as real-time PCR and microarray-based gene expression analysis.

Source -

Microchip-Based Solid-Phase Purification of RNA from Biological Samples
Having previously detailed a method for chip-based extraction of DNA (Anal. Chem. 2003, 75, 1880−1886.), we describe here a microchip-based solid-phase extraction method for purification of RNA from biological samples is demonstrated. The method involves the use of silica beads as a solid phase, and the capacity of the device containing silica beads for RNA, RNA in the presence of protein, and DNA was determined. The capacity of the device for RNA binding in the presence of protein is 360 ng, which demonstrates sufficient capacity of the device for complete genetic analysis. An extraction of RNA can be performed on the device in as few as 9 min (analytical time), a time comparable to that of a commercial extraction method, but with less reagent consumption. The microchip-based extraction is also performed in a closed system, unlike the commercial extraction method, which provides the advantage of decreased opportunity for the introduction of RNases and contaminantsessential for the sensitive RNA-based analyses presented in this work. RNA purified using the device was shown to be amplifiable using reverse transcription PCR (RT-PCR), allowing for translation of the method to the purification and subsequent amplification of biological samples. RNA was purified using the microchip-based method from neat semen, a mock semen stain, and cultured cells from a common pediatric cancer, alveolar rhabdomyosarcoma. Source -

Isolation and Detection of Enterovirus RNA
Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in largevolume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold

concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial realtime NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR. Source -

RNA isolation for gene expression analyses
The analysis of gene expression data in clinical medicine has been plagued by the lack of a critical evaluation of accepted methodologies for the collection, processing, and labeling of RNA. In the present report, the reliability of two commonly used techniques to isolate RNA from whole blood or its leukocyte compartment was compared by examining their reproducibility, variance, and signal-to-noise ratios. Whole blood was obtained from healthy subjects and was either untreated or stimulated ex vivo with Staphylococcus enterotoxin B (SEB). Blood samples were also obtained from trauma patients but were not stimulated with SEB ex vivo. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system or from isolated leukocytes. Biotin-labeled cRNA was hybridized to Affymetrix GeneChips. The Pearson correlation coefficient for gene expression measurements in replicates from healthy subjects with both techniques was excellent, exceeding 0.985. Unsupervised analyses, including hierarchical cluster analysis, however, revealed that the RNA isolation method resulted in greater differences in gene expression than stimulation with SEB or among different trauma patients. The intraclass correlation, a measure of signal-to-noise ratio, of the difference between SEB-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples than in whole blood: 0.75 vs. 0.46 (P = 0.002). At the P < 0.001 level of significance, twice as many probe sets discriminated between SEB-stimulated and unstimulated blood with leukocyte isolation than with PAXgene. The findings suggest that the method of RNA isolation from whole blood is a critical variable in the design of clinical studies using microarray analyses Source -

Isolation of RNA from Formalin-Fixed Paraffin-Embedded Tissues

Over the last decades, a large number of archival formalin-fixed paraffin-embedded (FFPE) tissue banks have been established worldwide. Based on the attached clinical and outcome information, these tissue banks have become an invaluable source for investigators in cancer research and other diseases. Traditionally, it was considered very difficult to perform RT-PCR using archival tissue samples, partly because of chemical modifiaction and degradation of RNA, partly because the amounts of RNA present were too small to be amplified by conventional means. However, improved techniques for extracting RNA from fixed specimens based on the use of proteinase K digestion followed by chromatography with silica-based spin columns, and the application of highly sensitive fluorescence-based real-time RT-PCR procedures have shown that it is possible to detect and quantify mRNA in FFPE tissues. The newly developed High Pure FFPE RNA Micro Kit addresses the increasing demand for RNA isolation from archived tissue samples. The kit provides an optimized solution for RNA extraction especially from thin tissue slices (1 µm–10 µm). It includes a simple, phenol-free workflow, an on-column DNase digestion, as well as a low elution volume for direct use of the isolated RNA in RT-PCR analysis. High Pure Micro Spin Column The High Pure Micro Spin Columns are designed for use in standard table-top centrifuges (Figure 1). In order to estimate the performance of the spin columns, a variable amount of pre-purified RNA was loaded onto the columns. The highest recovery was observed when 10.5 µg of total RNA were loaded onto the columns. Nevertheless, even for 42 µg of pre-purified total RNA a recovery of 75% was observed (Table 1). Also, the minimal elution volume was tested. Five micrograms of total RNA could be eluted with more than 80% recovery using 10 µl elution volume (Table 2). Based on the design of the volume-reducing collecting funnel, no residual buffers remain in the column after centrifugation, making this spin column easy to use by avoiding any carryover contamination in the eluted RNA. Isolation of Total RNA from FFPE Tissue Sections Isolation of RNA from FFPE material using the newly developed High Pure FFPE RNA Micro Kit was evaluated with four different tissue sections in comparison with kits offered by two competitors. Eluted RNA was quantified using a NanoDrop instrument (NanoDrop Technologies, USA) and loaded onto a Bioanalyzer instrument (Agilent, USA) in order to elucidate the RNA fragment length distribution within the different samples. Quality of the eluted RNA was further tested in a one-step standard RT-PCR assay by amplifying similar amounts of RNA in a 3 log dilution series. Crossing points observed were used for the interpretation of amplification efficiency/linearity as well as for comparison with kits from two commercial suppliers. Four different FFPE tissues were used for this analysis, and 5-µm slices were subjected to RNA isolation. For each kit and tissue, RNA isolation was performed in triplicate and the

mean yield is displayed in Table 3. The High Pure FFPE RNA Micro Kit shows a higher RNA yield for all tissues compared with supplier A as well as a higher RNA yield for 3 out of 4 tissues compared with supplier B. For one sample (xenograft) RNA isolation comepletely failed using the kit of supplier B. The quality of the RNA displayed by the 260/280 OD ratio was similar in all samples analyzed. The RNA fragment length distribution was analyzed on a Bioanalyzer instrument. We observed characteristic differences between the different RNA isolation kits, and a high degree of similarity among samples isolated with the same kit. The fragment length distribution of RNA isolated from the same tissue block using kits from three different suppliers is shown in Figure 2. Since the pico-chip format was used for the Bioanalyzer instrument, varying amounts of loaded RNA (1.3 ng–2.1 ng) were applied. All samples show RNA degradation based on the formalin fixation as well as the storage in paraffin blocks. Using the High Pure FFPE RNA Micro Kit, a good recovery of small fragments together with the preservation of longer fragments was observed. RNA isolated with the kit of supplier A shows a loss of small RNA fragments. RNA isolated with supplier B's kit shows a good recovery of small fragments as well as a decrease in the amount of longer fragments, indicating an additional RNA degradation during the isolation process. In order to evaluate the quality of the isolated RNA we performed a one-step RT-PCR assay on a 3 log dilution row. Four different RNA amounts (50 ng, 5 ng, 500 pg, 50 pg) of the same sample were subjected to RT-PCR analysis using a ß2-microtubulin specific PCR amplification with a LightCycler® 1.5 Instrument and SYBR Green I staining. Figure 3 displays the crossing point values of a ß2-microtubulin specific RT-PCR performed with a 3 log dilution row of three different RNA samples isolated from the same FFPE tissue block by three different RNA isolation kits. The samples subjected to RT-PCR were similar to those analyzed with the Bioanalyzer instrument (Figure 2). RNA isolated with the High Pure FFPE RNA Micro Kit showed a high amplification efficiency based on the slope of 3.172 for the 3 log dilution series as well as the lowest crossing point values compared with the other two kits. RNA isolated with a kit from supplier A displayed a high amplification efficiency (slope = -3.107) but the highest crossing point values. RNA isolated with a kit from supplier B had a slope of -2.8 and crossing point values between the RNA isolated with the High Pure FFPE RNA Micro Kit and supplier A's kit. Conclusions The new High Pure Micro Spin column format is ideal for isolating and purifying small sample amounts in a fast and convenient workflow. RNA purity and concentrations are suitable for direct use in RT-PCR applications. The High Pure FFPE RNA Micro Kit is optimized for ease of use, providing a fast and simple tool for isolation of RNA from thin (1 µm–10 µm) FFPE tissue slices. In comparison with the kits of two suppliers, the High Pure FFPE RNA Micro Kit displayed a superior performance based on high yields as well as optimal performance in a RT-PCR application shown by low crossing point values and a high linearity/amplification efficiency Source -

RNA Isolation from Yeast Using Silica Matrices
RNA isolation from yeast is complicated by the need to initially break the cell wall. While this can be accomplished by glass bead disruption or enzyme treatment, these approaches result in DNA contamination and/or the need for incubation periods. We have developed a protocol for the isolation of RNA samples from yeast that minimizes degradation by RNases and incorporates two purification steps: acid phenol extraction and binding to a silica matrix. The procedure requires no precipitation steps, facilitating automation, and can be completed in less than 90 min. The RNA quality is ideal for microarray analysis. Methods & results We have adapted the protocol from the NucleoSpin RNA II kit (Macherey-Nagel, Germany) to allow rapid cell lysis and increased RNA purification. Saccharomyces cerevisiae are grown in rich media to exponential phase (A600 nm approximately 2.0). The culture is placed on ice for 5 min. Five milliliters of yeast cells (approximately 108) are harvested by centrifugation at 1000 g and washed with ice-cold water. The cell pellet is resuspended in 300 μL of RA1 buffer, provided by the manufacturer, and 0.3 g of 500-μm acid-washed glass beads (BioSpec Products, Inc., Bartlesville, OK) is added. The cells are vortexed at high speed for 2 min. During this step, the glass beads break the cells and, although heating occurs, RNases are efficiently denatured by the guanidine contained in the RA1 buffer. Glass beads and cell debris are pelleted by centifugation at 16,000 g for 5 min at 4°C. The aqueous phase is then vortexed with 500 μL of acid phenol (Fisher Scientific) for 10 sec, and incubated on ice for 5 min. Two hundred microliters of chloroform (EM Science, Gibbstown, WV) is added and the aqueous phase collected after centrifugation at 16,000 g for 5 min at 4°C. The chloroform extraction is then repeated. The acid phenol extraction further ensures denaturation of RNases and eliminates DNA, which remains in the phenol phase.3 To complete the purification of the RNA, 300 μL of RNase-free 70% ethanol is added to the aqueous phase. This solution can then be applied directly to the silica matrix in the NucleoSpin RNA II kit. Further steps proceed as described by the manufacturer (MachereyNagel, Easton, PA). The matrix is desalted, and then DNA is removed by treatment with DNase. The matrix is washed and RNA eluted with 40–60 μL of water. Using this procedure, we have isolated 25–30 μg of RNA from 108 cells. The A260/A280 ratio for six samples averaged 2.0 ± 0.1, indicating that the samples were free of protein. Samples were also analyzed on an Agilent 2100 Bioanalyzer (Figure 11). Discussion: Based upon a rRNA [25S:18S] ratio of 1.9, we conclude that the RNA was intact. In addition, the RNA was found to be fully stable after storage at −70°C for 3 wks, after which it was efficiently labeled and used in microarray analyses. This approach for purification of RNA from Saccharomyces cerevisiae has several features that make it advantageous for microarray analysis. The cells are rapidly broken by glass beads, and RNases are quickly inactivated in guanidine. The extraction with acid phenol aids in the rapid elimination of protein and DNA, though incubation with hot phenol is not required as it is in other protocols.3,6 Use of the silica matrix allows for additional purification and concentration of the RNA sample under conditions in which any RNase is inactive. The need for reagents such as diethylpyrocarbonate to inactivate RNases is unnecessary, allowing

for maximal probe synthesis in subsequent steps. Finally, the protocol is rapid and allows efficient handling of multiple samples. In developing this protocol, we tried changing the order of the steps. In initial trials, phenol extraction preceded guanidine extraction. While efficient cell lysis did occur, binding to the silica matrix was seriously compromised. Source -

RNA-Based Drug Screening Using Automated RNA Purification and Realtime RT-PCR1
A new system has been developed for RNA-based drug screening, and the feasibility of this approach has been demonstrated by the identification of new immunomodulating compounds. Peripheral blood mononuclear cells were chosen as the cellular assay system. Cells were either stimulated by TPA/ionomycin to produce T cell cytokines as asthma targets or stimulated by lipopolysaccharide to produce proinflammatory cytokines as targets for chronic obstructive pulmonary disease (COPD). The authors developed a new fully automated system for RNA purification from cells grown in 96-well plates. Gene expression was determined in 384-well plates using real-time quantitative one-tube RT-PCR. Small interdonor variation could be demonstrated. The assay system was validated with known immunosuppressants cyclosporine and dexamethasone. Screening of 800 compounds resulted in 9.5% compounds inhibiting the induction of at least 1 T cell derived cytokine and 6.8% compounds inhibiting at least 1 cytokine relevant for COPD. All these compounds were retested by analyzing remaining RNA from the 1st round of screening. The reproducibility of hits was between 56% and 74% for different cytokines. One compound selectively inhibited TNF, which was confirmed by IC50 determination. Analyzing its effect on cells from different donors revealed little interdonor variation. In conclusion, the authors established fully automated RNA isolation and precise gene expression profiling using real-time RT-PCR for drug screening. Source -

RNA extraction

The reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis.


Frozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in -80°C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3'ends of mRNA motif, respectively.

Total yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptasepolymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed.

Our data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues. Source -

Shared By:
Tags: Kit.
Description: RNA Kit.