Message t7 ultra.0306.fm

mMESSAGE mMACHINE® T7 Ultra
Synthesis of Translation Enhanced Capped Transcripts
Catalog #1345
Protocol
version 0306
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Background
Synthesis of capped
Ambion’s mMESSAGE mMACHINE® T7 Ultra Kit (US patents issued and pending) is designed for the in vitro synthesis of large amounts of efficiently and correctly capped RNA. In addition, this kitincludes reagents for in vitro poly(A) tailing to make mRNA specifically for microinjections and trans-fections. mMESSAGE mMACHINE T7 Ultra reactions include a novel cap analog called Anti-ReverseCap Analog or ARCA (Stepinski J et al. 2001 and Peng et al. 2002). In ARCA, one of the 3' OHgroups (closer to 7MG) is eliminated from the cap analog and is substituted with –OCH (see Figure below). This modification allows T7 RNA polymerase to initiate transcription only with the remaining–OH group and thus synthesize RNAs capped exclusively in the correct orientation. Substitution oftraditional Cap Analog with ARCA allows for synthesis of capped RNAs that are 100% functional, incontrast to transcription reactions using traditional cap analog where only half of the cap analog isincorporated in the correct orientation. GTP and ARCA are supplied in a single solution with anARCA:GTP ratio of 4:1, which is optimal for maximizing both RNA yield and the proportion ofcapped transcripts.
Figure 1. ARCA Structure
The high yields are achieved by optimizing reaction conditions for RNA synthesis in the presence ofhigh nucleotide concentrations (U.S. patents issued and pending). The RNA produced is protectedfrom degradation by contaminating ribonucleases with SUPERase•In™ (U.S. and foreign patents pend-ing), a broad spectrum RNase inhibitor present in the mMESSAGE mMACHINE T7 Ultra EnzymeMix. The mMESSAGE mMACHINE T7 Ultra Kit contains all the buffers and reagents necessary forten 20 µl transcription reactions. Using the control template supplied with the kit (Xenopus elongationfactor 1α, pTRI Xef), each mMESSAGE mMACHINE T7 Ultra reaction will yield approximately20–30 µg of RNA.
Poly(A) tailing of
Poly(A) tailing reagents are also included with the mMESSAGE mMACHINE T7 Ultra Kit; they can be used to add a ≥50–100 base poly(A) tail to the RNA transcripts. This is accomplished using E. coliPoly(A) Polymerase (E-PAP) and ATP. The resulting capped and tailed RNA can then be used in trans-fection or microinjection experiments where enhanced translation over untailed mRNAs may be seendue to increased mRNA stability and translation efficiency (Bernstein and Ross 1989, Gallie 1991,Harland and Misher 1989, Khaleghpour et al. 2001).
Storage and Stability
The mMESSAGE mMACHINE T7 Ultra Kit should be stored at –20°C in a non frost-free freezer.
Keep all reagents on ice while using the kit; the nucleotides and enzymes (T7 Enzyme Mix and E-PAP)are especially labile. Properly stored kits are guaranteed for 6 months from the date received.
Ambion Inc. • USA: 800-888-8804 • Can: 800-445-1161 • Intl: +1-512-651-0200 • • Ambion (Europe) Ltd. • UK: 08001 381 836 • DE: 08001 813 273 • CH: 0800 837 122 • mMESSAGE mMACHINE® T7 Ultra
Protocol
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Materials Provided with the Kit
Reagents are included for 10 mMESSAGE mMACHINE T7 Ultra reactions.
Transcription and capping components:
Component
Buffered 50% glycerol containing RNA polymerase,SUPERase•In, and other components salts, buffer, dithiothreitol, and other ingredients a neutralized buffered solution containing: Poly(A) tailing components:
Component
Materials Not Provided with the Kit
• DNA template: The DNA template must have the T7 RNA polymerase promoter site upstream of the sequence to be transcribed. The suggested template concentration is 0.5 µg/µl in dH O or TE (10 mM Tris-HCl (pH 7–8), 1 mM EDTA).
• (optional) Labeled nucleotide(s): [α-32P]UTP or [α-32P]CTP can be used as a tracer for transcription and [α-32P]ATP can be used as a tracer to assess the poly(A) tailing reaction. Any specific activity isacceptable.
• (optional) For purification of the synthesized RNA: Buffer- or water-saturated phenol/chloroformIsopropanolSpin Columns Ambion Inc. • USA: 800-888-8804 • Can: 800-445-1161 • Intl: +1-512-651-0200 • • • • Ambion (Europe) Ltd. • UK: 08001 381 836 • DE: 08001 813 273 • CH: 0800 837 122 • • • • mMESSAGE mMACHINE® T7 Ultra
Protocol
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Related Products Available from Ambion
MEGAclear™
MEGAclear purifies RNA from mMESSAGE mMACHINE T7 Ultra, and other enzymatic reactions yielding high quality RNA free of unincorporated nucleotides, enzymes and buffer components with close to 100%recovery of input RNA.
RNase-free Tubes & Tips
Ambion’s RNase-free tubes and tips are available in most commonly used sizes and styles. They are guaranteed RNase- and DNase-free. See our latest catalog for specific information.
RNaseZap®
RNase Decontamination Solution. RNaseZap is simply sprayed or poured onto surfaces to instantly inactivate RNases. Rinsing twice with water will eliminate all traces of RNase and RNaseZap.
NucAway™ Spin Columns
Guaranteed RNase- and DNase-free, Ambion’s NucAway Spin Columns provide a fast, efficient way to remove unincorporated nucleotides, and to effect buffer exchange after probe synthesis and other reactions.
RNA Storage Solutions
Three different choices for safe, RNase-free resuspension of RNA pellets. Choose one or more of the following: THE RNA Storage Solution, Cat. #’s 7000, 70010.1 mM EDTA Cat. #’s 9911, 9912TE Buffer, Cat. #’s 9860, 9861 High concentration SP6, T7
Cloned, high purity RNA polymerases. These RNA polymerases are rigorously tested for superior performance, and T3 RNA Polymerases
DNA-free™
DNase treatment and removal reagents. This product contains Ambion’s ultra-high quality RNase-free DNase 1 and reaction buffer for degrading DNA. It is ideal for removing contaminating DNA from RNA preparations. Anovel reagent for removing the DNase without the hassles or hazards of phenol extraction or alcohol precipita-tion is also included.
Electrophoresis Reagents
Ambion offers gel loading solutions, agaroses, acrylamide solutions, powdered gel buffer mixes, nuclease-free water, and RNA and DNA molecular weight markers for electrophoresis. Please see our catalog for a completelisting as this product line is always growing.
Proteinase K
Proteinase K is a non-specific serine protease commonly used in molecular biology to remove protein contami- nants from nucleic acids. Ambion supplies Proteinase K in powder form, and as a 50% glycerol solution.
Ambion offers a full line of prepared phenol solutions for most molecular biology needs. These premixed, qual- ity-tested, saturated phenols are ready-to-use and eliminate the handling concerns associated with preparingphenol for use from solid phenol.
Preparation of Template DNA
Linearized plasmid DNA and PCR products that contain a T7 RNA polymerase promoter site can beused as templates for in vitro transcription with mMESSAGE mMACHINE T7 Ultra. In general, anyDNA with a T7 promoter site, that is pure enough to be easily digested with restriction enzymes can beused for in vitro transcription.
Figure 2. T7 polymerase promoter: minimal sequence requirements
TAATACGACTCACTATAGGGAGA
The +1 base (in bold) is the first base incorporated into RNA during tran-scription. The underline shows the minimum promoter sequence needed for efficient transcription.
Template size
The mMESSAGE mMACHINE T7 Ultra kit is designed to function best with templates that code for
RNA transcripts in the 0.3 to 5 kb range. The kit can be used to produce longer, or shorter RNA.
Ambion Inc. • USA: 800-888-8804 • Can: 800-445-1161 • Intl: +1-512-651-0200 • • • • Ambion (Europe) Ltd. • UK: 08001 381 836 • DE: 08001 813 273 • CH: 0800 837 122 • • • • mMESSAGE mMACHINE® T7 Ultra
Protocol
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Orientation
If sense RNA is needed, it is important to transcribe using the RNA polymerase corresponding to the
phage promoter at the 5', or amino-terminal side of the coding region of the protein (using
promoter 1 in the diagram below). If the template consists of a plasmid, it should be linearized in the
polylinker at the opposite (3' or carboxy-terminal side) of the protein-coding region.
Antisense (mRNA-complementary) transcripts will be synthesized if the RNA polymerase correspond-
ing to the RNA phage promoter at the 3', or carboxy-terminal side of the coding region of the protein
is used (using promoter 2 in the diagram below).
Transcription using the RNA polymerase corresponding to promoter 1 willmake sense RNA (the same sequence as the mRNA). If the RNA poly-merase for promoter 2 is used, antisense RNA will be transcribed.
PCR templates
DNA generated by PCR can be transcribed directly from the PCR provided it contains a T7 RNAPolymerase promoter upstream of the sequence to be transcribed. PCR products should be examinedon an agarose gel before use as a template in mMESSAGE mMACHINE T7 Ultra to estimate concen-tration, and to verify that the products are unique, and the expected size. Plasmid Templates
DNA should be relatively free of contaminating proteins and RNA. We observe the greatest yields withvery clean template preparations. Most commercially available plasmid preparation systems yield DNAthat works well in the mMESSAGE mMACHINE T7 Ultra Kit.
Linearization
Plasmid DNA must be linearized with a restriction enzyme downstream of the insert to be tran-scribed. Circular plasmid templates will generate extremely long, heterogeneous RNA transcriptsbecause RNA polymerases are very processive. It is generally worthwhile to examine the linearizedtemplate DNA on a gel to confirm that cleavage is complete. Since initiation of transcription is oneof the limiting steps of in vitro transcription reactions, even a small amount of circular plasmid in atemplate prep will generate a large proportion of transcript.
Although we routinely use all types of restriction enzymes, there has been one report of low leveltranscription from the inappropriate template strand in plasmids cut with restriction enzymes leav-ing 3' overhanging ends (produced by Kpn I, Pst I, etc.; Schendorn and Mierindorf, 1985). After linearization
Terminate the restriction digest by adding the following: • 1/10th volume of 3 M NaOAc or 5 M NH OAc
• 2 volumes of ethanolMix well and chill at –20°C for at least 15 min. Then pellet the DNA for 15 min in a microcentri-fuge at top speed. Remove the supernatant, re-spin the tube for a few seconds, and remove theresidual fluid with a very fine-tipped pipet. Resuspend in dH O or TE buffer at a concentration of Note that DNA from some miniprep procedures may be contaminated with residual RNase A. Also,restriction enzymes occasionally introduce RNase or other inhibitors of transcription. When tran-scription from a template is suboptimal, it is often helpful to treat the template DNA withproteinase K (100–200 µg/ml) and 0.5% SDS for 30 min at 50°C, follow this with phenol/chloro-form extraction (using an equal volume) and ethanol precipitation.
Ambion Inc. • USA: 800-888-8804 • Can: 800-445-1161 • Intl: +1-512-651-0200 • • • • Ambion (Europe) Ltd. • UK: 08001 381 836 • DE: 08001 813 273 • CH: 0800 837 122 • • • • mMESSAGE mMACHINE® T7 Ultra
Protocol
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Capped Transcription Reaction Assembly
1. Thaw the frozen
Place the RNA Polymerase Enzyme Mix on ice, it is stored in glycerol and will not be frozen at –20°C. reagents
Vortex the 10X T7 Reaction Buffer and the T7 2X NTP/ ARCA until they are completely in solution.
Once thawed, store the ribonucleotides T7 (2X NTP/ARCA) on ice, but keep the 10X T7 Reaction
Buffer at room temperature while assembling the reaction.
All reagents should be microfuged briefly before opening to prevent loss and/or contamination ofmaterial that may be present around the rim of the tube.
2. Assemble
The spermidine in the 10X T7 Reaction Buffer can coprecipitate the template DNA if the reaction is transcription
reaction at room
Add the 10X T7 Reaction Buffer after the water and the ribonucleotides are already in the tube.
temperature
The following amounts are for a single 20 µl reaction. Reactions may be scaled up or down if desired.
The following reaction setup is recommended when the RNA produced will be 300 bases to 5 kb in length. Component
3. Mix thoroughly
Gently flick the tube or pipette the mixture up and down gently, and then microfuge tube briefly tocollect the reaction mixture at the bottom of the tube.
4. Incubate at 37°C,
Typically, 80% yield is achieved after a 1 hour incubation. For maximum yield, we recommend a 2 hour A second hour of incubation is recommended for synthesis of <300 base transcripts and for inefficientlytranscribed templates.
If the transcription reaction is trace-labeled:
After the incubation (before or after DNase treatment), remove an aliquot of trace-radiolabeledreactions to assess yield by TCA precipitation (see section 5. Add 1 µl DNase I,
This DNase treatment removes the template DNA. For many applications it may not be necessary mix well and
because the template DNA will be present at a very low concentration relative to the RNA.
incubate 15 min at
b. Incubate at 37°C for 15 minutes.
H. Poly(A) Tailing Protocol
1. Add tailing reagents
Add the tailing reagents to the 20 µl mMESSAGE mMACHINE T7 Ultra reaction in the following order: Amount Component
(20 µl) mMESSAGE mMACHINE T7 Ultra reaction 36 µl Nuclease-free Water20 µl 5X E-PAP Buffer10 µl 25 mM MnCl210 µl ATP Solution Ambion Inc. • USA: 800-888-8804 • Can: 800-445-1161 • Intl: +1-512-651-0200 • • • • Ambion (Europe) Ltd. • UK: 08001 381 836 • DE: 08001 813 273 • CH: 0800 837 122 • • • • mMESSAGE mMACHINE® T7 Ultra
Protocol
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2. Reserve 2.5 µl of the
Remove 2.5 µl of the reaction mixture before adding the E-PAP enzyme; this minus-enzyme control reaction mixture
will be run on a gel next to the tailed RNA at the end of the experiment.
3. Add 4 µl E-PAP, mix
Add 4 µl of E-PAP, and mix gently.
4. Incubate at 37°C for
After the 30–45 min incubation at 37°C, place the reaction on ice.
30–45 min
If the poly(A) tailing reaction is trace-labeled:
After the incubation (before RNA recovery), remove an aliquot of trace-radiolabeled reactions toassess yield by TCA precipitation (see section .
Recovery of the RNA
The degree of purification required after the transcription reaction depends on what will be done with theRNA. Four different methods follow, choose one or more according to your application and resources.
1. Ambion’s
MEGAclear was developed specifically for purifying RNA from high yield in vitro transcription reac- MEGAclear™
tions. The quick and simple procedure removes nucleotides, short oligonucleotides, proteins, and saltsfrom RNA. The RNA recovered can be used for any application that requires high purity RNA.
2. Lithium chloride
Lithium Chloride (LiCl) precipitation is a convenient and effective way to remove unincorporated precipitation
nucleotides and most proteins. Lithium chloride precipitation, however, does not precipitate transferRNA and may not efficiently precipitate RNAs smaller than 300 nucleotides. Also, the concentration ofRNA should be at least 0.1 µg/µl to assure efficient precipitation. To precipitate from mMESSAGEmMACHINE T7 Ultra reactions that are thought to have relatively low yields of RNA, do not dilutethe transcription reaction with water prior to adding the LiCl Precipitation Solution in step low. a. Stop the reaction and precipitate the RNA by adding 50 µl LiCl Precipitation Solution. b. Mix thoroughly. Chill for ≥30 min at –20°C. c. Centrifuge at 4°C for 15 min at maximum speed to pellet the RNA. d. Carefully remove the supernatant. Wash the pellet once with ~1 ml 70% ethanol, and re-centrifuge to maximize removal of unincorporated nucleotides.
e. Carefully remove the 70% ethanol, and resuspend the RNA in a solution or buffer* appropriate for your application. Determine the RNA concentration and store frozen at –20°C or –70°C.
3. Spin column
Spin columns will remove unincorporated nucleotides, including unincorporated ARCA that may chromatography
Prepared spin columns such as Ambion’s NucAway™ Spin Columns can be used by following the man-ufacturer’s instructions.
4. Phenol:chloroform
This is the most rigorous method for purifying transcripts. It will remove all enzyme and most of the extraction and
free nucleotides from mMESSAGE mMACHINE T7 Ultra reactions. Since the RNA is precipitated, isopropanol
this method can also be used for buffer exchange.
precipitation
a. Add 10 µl Ammonium Acetate Stop Solution, and mix thoroughly.
b. Extract with an equal volume of phenol/chloroform (it can be water-saturated, buffer-saturated, or acidic), and then with an equal volume of chloroform.
(Optional: back-extract the organic phase with 50 µl dH O.) Ambion offers several products for RNA storage, these include: Nuclease-free Water (not DEPC-treated): Cat #9930–9934THE RNA Storage Solution: Cat #7000, 7001TE Buffer: Cat #9860, 98610.1mM EDTA: Cat #9911, 9912 Ambion Inc. • USA: 800-888-8804 • Can: 800-445-1161 • Intl: +1-512-651-0200 • • • • Ambion (Europe) Ltd. • UK: 08001 381 836 • DE: 08001 813 273 • CH: 0800 837 122 • • • • mMESSAGE mMACHINE® T7 Ultra
Protocol
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c. Precipitate the RNA by adding 1 volume of isopropanol and mixing well.
d. Chill the mixture for at least 15 minutes at –20°C. Centrifuge at 4°C for 15 minutes at maximum speed to pellet the RNA. Carefully remove the supernatant solution and resuspend the RNA in asolution or buffe appropriate for your application.
e. Store frozen at –20°C or –70°C.
Quantitation of Reaction Products
1. Quantitation by UV
Reading the A of a diluted aliquot of the reaction is clearly the simplest way to determine yield, but light absorbance
any unincorporated nucleotides and/or template DNA in the mixture will contribute to the reading.
Typically, a 1:100 dilution of an aliquot of a mMESSAGE mMACHINE T7 Ultra reaction will give anabsorbance reading in the linear range of a spectrophotometer. For single-stranded RNA, 1 A unit corresponds to 40 µg/ml, so the RNA yield can be calculated as 2. Assessing RNA yield
If you have a fluorometer, or a fluorescence microplate reader, Molecular Probes’ RiboGreen® fluores- with RiboGreen®
cence-based assay for RNA quantitation is a convenient and sensitive way to measure RNA concentra-tion. Follow the manufacturer’s instructions for using RiboGreen.
3. Quantitation by
The intensity of ethidium bromide staining can be used to get a rough estimation of the RNA yield. ethidium bromide
fluorescence
a. Ethidium bromide spot assay
If unincorporated nucleotides have been removed, an ethidium bromide spot assay can be used toquantitate RNA concentration. Make a standard curve with several 2 fold dilutions of an RNA solu-tion of known concentration. Start at about 80 ng/µl, and go down to about 1.25 ng/µl. Make afew dilutions of the unknown RNA, and add ethidium bromide to 1 ng/µl to each dilution of bothRNAs. Spot 2 µl of the standard curve RNA samples and the unknown RNA dilutions onto plasticwrap placed on a UV transilluminator. Compare the fluorescence of the RNAs to estimate the con-centration of the unknown RNA sample. Make sure that the sample dilutions are in the linear rangeof ethidium bromide fluorescence. This assay will detect as little as 5 ng of RNA with an error ofabout 2 fold.
b. Denaturing gel electrophoresis
If unincorporated nucleotides have not been removed from the reaction, an aliquot of the mMES-SAGE mMACHINE T7 Ultra reaction should be run on a denaturing agarose gel alongside an ali-quot of an RNA of known concentration. Stain the samples with ethidium bromide, and simplycompare the intensity of the unknown sample to the known RNA to estimate its concentration.
4. Quantitation by
mMESSAGE mMACHINE T7 Ultra reactions can be trace radiolabeled during either the transcription trace radiolabeling
reaction or the poly(A) tailing reaction. Trace labeling the transcription reaction makes it possible toaccurately determine the transcript yield; trace labeling the poly(A) tailing reaction enables determina-tion of the amount of ATP incorporated into the poly(A) tail.
• To determine RNA yield from a trace labeled transcription reaction, TCA precipitate an aliquot of the reaction as described in section elow, and use the results to calculate RNA yield as describedin section • Trace labeling the poly(A) tailing reaction will provide an indication of how well the tailing reaction worked. To trace label poly(A) tailing reactions, simply add 1 µl of [α-32P]ATP (any specific activitycan be used) to the tailing reaction at step After the reaction iscomplete, TCA precipitate an aliquot of the reaction as described in section elow. Successful reactions incorporate 50% or more radiolabel.
Ambion Inc. • USA: 800-888-8804 • Can: 800-445-1161 • Intl: +1-512-651-0200 • • • • Ambion (Europe) Ltd. • UK: 08001 381 836 • DE: 08001 813 273 • CH: 0800 837 122 • • • • mMESSAGE mMACHINE® T7 Ultra
Protocol
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a. TCA precipitation
Dilute 5 µl of the completed mMESSAGE mMACHINE T7 Ultra reaction with 5 µl TE Buffer in a nuclease-free 1.5 ml microfuge tube, and vortex thoroughly to ensure that the newly synthesized RNA is completely solubilized.
ii. Add 150 µl of carrier DNA or RNA (1 mg/ml) (Ambion’s Sheared Salmon Sperm DNA Cat #9680 can be used for this) to the diluted reaction products, and mix thoroughly.
iii. Transfer 50 µl of the RNA + carrier nucleic acid mixture to aqueous scintillation cocktail and count in a scintillation counter. This will measure the total amount of radiolabel present in the reaction mixture (unincorporated and incorporated counts).
iv. Transfer another 50 µl of the RNA + carrier nucleic acid mixture to a 12 x 75 mm glass tube, and add 2 ml of cold 10% TCA (trichloroacetic acid). Mix thoroughly and place on ice for 10 minutes. This will precipitate nucleic acids, but not free nucleotides.
v. Collect the precipitate via vacuum filtration through a Whatman GF/C glass fiber filter (or its vi. Rinse the tube twice with 1 ml of 10% TCA and then rinse once with 3–5 ml of 95% ethanol. Pass each of the rinses through the GF/C filter.
vii. Place the filter in a scintillation vial, add aqueous scintillation cocktail, and count in a scintillation counter. This will give the TCA precipitated counts (radiolabel that was incorporated into RNA).
viii.Divide the cpm in Stepthe cpm in Stepo determine the fraction of label incorporated into RNA (multiply by 100 for percent incorporation).
b. Calculation of transcription reaction yield
Once the percent incorporation of radiolabel is known, it can be used to calculate the mass yield ofRNA transcribed in the mMESSAGE mMACHINE T7 Ultra reaction. The concentration of GTPlimits the amount of RNA that can be synthesized. For any tracer other than labeled GTP (e.g.
[α-32P] UTP), each 1% incorporation corresponds to about 2 µg of RNA synthesized.
In a mMESSAGE mMACHINE T7 Ultra reaction, if all four nucleotides are incorporated equally,39.6 µg of RNA will be produced if all of the 1.5 mM of GTP is incorporated into RNA (the sum ofthe molecular masses of the 4 nucleotides in RNA is about 1320). Since the ratio of cap analog toGTP is 4:1, this represents a maximal theoretical incorporation of 20% of the label. Ambion Inc. • USA: 800-888-8804 • Can: 800-445-1161 • Intl: +1-512-651-0200 • • • • Ambion (Europe) Ltd. • UK: 08001 381 836 • DE: 08001 813 273 • CH: 0800 837 122 • • • •

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