AmpliScribe T 7 Aminoallyl-RNA Transcription Kit

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1 Cat. No. AA50125 The AmpliScribe T7 Aminoallyl-RNA Transcription Kit enables high-yield production of aminoallyl-labeled RNA. The kit utilizes Epicentre s high yielding AmpliScribe T7-Flash in vitro transcription technology to incorporate 5-(3-aminoallyl)-UTP (AA-UTP) into the RNA transcript. Once purified, the aminoallyl-rna can be conjugated to the amine-reactive N-hydroxy succinamidyl (NHS) esters of biotin (e.g., Biotin-X-X-NHS) or fluorescent-nhs dyes (e.g., Cy -NHS dyes) for use in microarray, in situ hybridization, blotting or other studies utilizing a non-radioactive-labeled RNA. The in vitro transcription reaction conditions have been optimized to produce the highest yield of aminoallyl-rna for high signal intensity after biotin or fluorescent dye conjugation. Applications Production of non-radioactive RNA probes for: DNA microarray experiments. In situ hybridization experiments. Blotting experiments. Desc. Concentration Quantity AmpliScribe T7 Aminoallyl-RNA Transcription Kit Contents The AmpliScribe T7 Aminoallyl-RNA Transcription Kit contains sufficient reagents to perform 25 reactions. AmpliScribe T7-Flash Enzyme Solution 50 μl 100 mm ATP, CTP, and GTP Solutions each at 50 μl 100 mm UTP Solution 35 μl 50 mm Aminoallyl-UTP Solution 35 μl AmpliScribe T7-Flash 10X Reaction Buffer 50 μl 100 mm Dithiothreitol (DTT) 50 μl RiboGuard RNase Inhibitor 15 μl RNase-Free Water 1 ml Control Template 0.5 μg/μl 10 μl RNase-Free DNase 1 MBU/μl 25 μl Lit. # 227 6/2011 1

2 Product Specifications Storage: Store only at 20 C in a freezer without a defrost cycle. Do not store at 70 C. Contaminating Activity Assays: All of the components of the AmpliScribe T7 Aminoallyl-RNA Transcription Kit are free of detectable RNase activity, and all of the components except DNase I are free of detectable exo- and endonuclease activities. Control Template: The control template is a 4.2-kb linearized plasmid, containing a 1.4-kb lambda DNA insert, that will produce a 1,380-b runoff transcript. DNase I Unit Definition: 1 Molecular Biology Unit (MBU) of DNase I digests 1 microgram of puc19 DNA to oligodeoxynucleotides in 10 minutes at 37 C. Related Products: The following products are also available: TargetAmp Aminoallyl-aRNA Amplification Kits AmpliScribe T7-Flash Biotin-RNA Transcription Kit AmpliScribe T7-Flash Transcription Kit Aminoallyl-UTP Solution Biotin-X-X-NHS RiboGuard RNase Inhibitor Notes on Using the AmpliScribe T7 Aminoallyl-RNA Transcription Kit 1. Template Preparation: Transcription templates should be linear double-stranded DNA with blunt or 5 -protruding ends. Templates containing 3 -protruding ends can produce spurious transcripts due to non-specific initiation. PCR products and cdna can also be used as templates, provided that the appropriate promoter has been incorporated into one of the primers used (e.g., a cdna template produced during an Eberwine RNA amplification reaction; see also Note 4). The quality of the DNA template directly affects the quantity and quality of the RNA produced. Generally, DNA is of sufficient quality for use if it is free of contaminating RNase and can be fully digested with restriction enzymes. To confirm that a template is fully linearized and intact, examine the DNA on an ethidium-stained agarose or polyacrylamide gel prior to use. Templates that give low yields or less than full-length transcripts may contain RNase or other contaminants. Such templates will usually give better results after the following treatment: a) Add Proteinase K to μg/ml and SDS to 0.5%. b) Incubate for minutes at 37 C. c) Extract with an equal volume of a 1:1 mixture of TE-saturated phenol/chloroform. d) Ethanol precipitate. e) Gently remove the supernatant and rinse the pellet with 70% ethanol. f ) Resuspend at 1.0 μg/μl in RNase-Free T 10 E 1 (10 mm Tris-HCl [ph 7.5], 1 mm EDTA). 2

3 2. Template Efficiency: Linearized plasmid templates cdnas and PCR product templates which produce transcripts of equivalent sizes are utilized with equal efficiency by the AmpliScribe T7 Aminoallyl-RNA Transcription Kit. The Control DNA Template produces greater than 160 μg of ~1.4-kb aminoallyl-rna per 1 μg of DNA template in a standard 20-μl reaction. Different templates may give different yields. Lower yields from an experimental template could be due to: a) Quality of template prep: Degraded templates, RNase or contaminants such as phenol, trace metals, and SDS may reduce yields. b) Transcriptional efficiency: Different templates may be transcribed more or less efficiently based on promoter strength, reinitiation rate, and termination efficiency. c) Size of the template: Yields may also differ based on the size of the template. 3. RNA Yield and the Amount of Plasmid DNA Template: The standard 20-μl, 30-minute reaction is optimized for transcription using 1 μg of linear DNA template, however, higher or lower amounts of DNA template can be used successfully. Table 1 summarizes our experiences with varying the amount of control DNA template in a standard AmpliScribe T7 Aminoallyl-RNA Transcription reaction. Results may vary depending on the template used. Increasing the reaction time for lesser amounts of template may increase the yield of RNA. Table 1. Yield of Aminoallyl-RNA from varying amounts of control template DNA from a standard 37 C, 20-μl AmpliScribe T7 Aminoallyl-RNA Transcription reaction over time. Results may vary depending on the template used. Template DNA (ng) Incubation Time (minutes) μg μg μg >100 μg μg μg μg μg μg μg μg μg μg μg μg >160 μg 1000 >160 μg >160 μg >160 μg >160 μg 4. Transcribing Aminoallyl-RNA for DNA Microarrays: The AmpliScribe T7 Aminoallyl- RNA Transcription Kit offers an efficient and cost effective alternative for producing labeled target RNA for use in DNA microarrays compared to in vitro transcription methods that rely on direct incorporation of a biotin- or fluorescent-ntp. Typically, in this application, the Poly(A) RNA (mrna) contained in a total cellular RNA sample is converted to cdna containing a phage T7 transcription promoter. The cdna is then transcribed, in the presence of a biotin- or fluorescent-ntp, to produce labeled RNA. (800)

4 If using the AmpliScribe T7 Aminoallyl-RNA Transcription Kit to label RNA for microarray use, it is important to consider the Poly(A) content of the total RNA sample used. For example, 5 μg of a total cellular RNA sample containing 2% Poly(A) RNA contains just 100 ng of Poly(A) RNA. If 100% of the Poly(A) RNA is converted to cdna transcription template, then the in vitro transcription reaction will contain just 100 ng of template and the transcription reaction time should be adjusted accordingly (refer to Table 1) to maximize the yield of the aminoallyl-rna. 5. Reaction Assembly: Assemble an AmpliScribe T7 Aminoallyl-RNA transcription reaction at room temperature! Assembly of the reaction at temperatures less than 22 C can result in formation of an insoluble precipitate. Storing the AmpliScribe T7- Flash 10X Reaction Buffer at 70 C may result in the formation of a white precipitate. If this happens, heat the tube to 37 C for 5 minutes and mix thoroughly to resuspend the precipitate. 6. Maintaining an RNase-free Environment: The RiboGuard RNase Inhibitor is a potent RNase inhibitor. However, creating an RNase-free work environment and maintaining RNase-free solutions is critical for performing successful in vitro transcription reactions. Therefore, we strongly recommend that the user: Autoclave all tubes and pipette tips that will be used in the RNA amplification reactions. Always wear gloves when handling samples containing RNA. Change gloves frequently especially after touching potential sources of RNase contamination such as door knobs, pens, pencils, and human skin. Always wear gloves when handling kit components. Do not pick up any kit component with an ungloved hand. Keep all kit components tightly sealed when not in use. Keep all tubes containing RNA tightly sealed during the incubation steps. Standard AmpliScribe T7 Aminoallyl-RNA Transcription Reaction We recommend that you perform all reactions in sterile 0.2 to 0.5-ml thin-walled tubes using sterile pipette tips and recently calibrated pipettors. Wear gloves when handling all kit components and reaction tubes. 1. Place the AmpliScribe T7-Flash Enzyme Solution on ice. Thaw the remaining in vitro transcription reagents at room temperature. Important! If a precipitate is visible in the thawed AmpliScribe T7-Flash 10X Reaction Buffer, heat the Buffer to 37 C until it dissolves. Keep the AmpliScribe T7-Flash 10X Reaction Buffer at room temperature. 2. Thoroughly mix the thawed AmpliScribe T7-Flash 10X Reaction Buffer. 4

5 3. Combine the following reaction components at room temperature in the order given. (see Note 5) x μl RNase-Free water μg linearized template DNA 2 μl AmpliScribe T7-Flash 10X Reaction Buffer 1.8 μl 100 mm ATP 1.8 μl 100 mm CTP 1.8 μl 100 mm GTP 1.2 μl 100 mm UTP 1.2 μl 50 mm Aminoallyl-UTP 2 μl 100 mm DTT 0.5 μl RiboGuard RNase Inhibitor 2 μl AmpliScribe T7-Flash Enzyme Solution 20 μl Total reaction volume 4. Incubate at 37 C for 30 minutes. Refer to Table 1 to determine the reaction time that will maximize the yield of aminoallyl-rna from the amount of template in the reaction. 5. Optional: If removal of the DNA template is desired, add 1 μl (1 MBU) of RNase-Free DNase I to the standard 20-μl reaction and incubate for 15 minutes at 37 C. Purifying the Aminoallyl-RNA The aminoallyl-rna can be purified by phenol extraction followed by ethanol precipitation or by spin column chromatography using, for example, a Qiagen RNeasy Mini column. Quantifying the Concentration and Yield of the Aminoallyl-aRNA Concentration and yield: Due to the high yield of aminoallyl-rna (AA-RNA) that is produced, the yield and concentration of AA-RNA can be determined easily and rapidly by UV spectroscopy. 1. Prepare a dilution of the AA-RNA into the minimum volume of water or TE Buffer required by the spectrophotometer cuvette that will be used. 2. Zero the spectrophotometer at 260 nm using the diluents (water or TE buffer) that was used to dilute the AA-RNA sample. 3. Measure and record the absorbance of the diluted AA-RNA at 260 nm (A 260 ). 4. Calculate the concentration of the AA-RNA. Use the conversion factor that an A 260 reading of 1.0 is equal to an RNA concentration of 40 μg/ml. AA-RNA concentration = (A 260 reading) x (dilution factor) x (40 μg/ml). Example: Dilution for A 260 measurement = 1:200 with an A 260 of the 1:200 dilution = AA-RNA concentration = (0.20) x (200) x (40 μg/ml) = 1600 μg/ml = (1.6 μg/μl) AA-RNA. (800)

6 5. Calculate the yield of AA-RNA using the formula: Yield of AA-RNA = (AA-RNA Concentration) x (Volume of AA-RNA). Example: 100 μl of AA-RNA recovered, 1.6 μg/μl AA-RNA determined above. AA-RNA yield = (1.6 μg/μl) x (100 μl) = 160 μg of AA-RNA. Labeling the Aminoallyl-RNA with Biotin-NHS or Fluorescent Dye-NHS The aminoallyl-rna produced using the kit can be readily labeled with amine-reactive N-hydroxysuccinimide (NHS) ester of Biotin (e.g., Biotin-X-X-NHS) or fluorescent dyes (e.g., Cy 3-NHS or Cy 5-NHS; GE Healthcare). In aqueous solution, the -NHS group of the biotin or fluorescent dye rapidly and with high efficiency reacts with the amine groups of the aminoallyl-uridine that was incorporated into the aminoallyl-rna during the in vitro transcription reaction. The end product is RNA with covalently attached biotin or fluorescent molecules. Biotin-X-X-NHS is now available from Epicentre in convenient 2.5 mg packages. The Biotin-X-X-NHS is provided in septum-sealed vials to prevent moisture from entering the vial. A complete protocol for conjugating the Biotin-X-X-NHS ester to aminoallyl-rna is provided with the product. AmpliScribe, RiboGuard, TargetAmp, and T7-Flash are trademarks of Epicentre, Madison, Wisconsin. RNeasy is a registered trademark of Qiagen Inc., Valencia, California. Cy is a trademark of Amersham Biosciences, Piscataway, New Jersey. Visit our technical blog: epicentral.blogspot.com 6

7 Notes (800)

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