Superscript Firststrand Synth cDNA Manual

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    Instruction Manual

    SuperScript First-Strand SynthesisSystem for RT-PCR

    Catalog No. 11904-018

    Version E

    11 August 2003

    53034

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    iii

    Table of Contents

    Kit Contents and Storage.......................................................................... 1

    Introduction ..............................................................................................2

    First-Strand cDNA Synthesis ................................................................... 5Amplification of the Target cDNA........................................................... 8

    Troubleshooting...................................................................................... 11

    Troubleshooting with the Control RNA .................................................13

    Additional Protocols ............................................................................... 16

    References ..............................................................................................20

    Related Products..................................................................................... 21

    Purchaser Notification ............................................................................ 23Technical Service ................................................................................... 25

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    1

    Kit Contents and Storage

    KitComponents

    The components of the SuperScript First-Strand Synthesis System

    for RT-PCR are listed below. Components are provided in

    sufficient quantities to perform a total of 50 separate reactions; each

    converts from 1 ng up to 5 g of total RNA into first-strand cDNA.

    The SuperScript First-Strand Synthesis System for RT-PCR is

    stored at 20C.

    Component Amount

    Oligo(dT)12-18 (0.5 g/l) 50 l

    Random hexamers (50 ng/l) 250 l

    10X RT buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl ] 1 ml

    25 mM MgCl2 500 l

    0.1 M DTT 250 l

    10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP) 250 l

    SuperScript II RT (50 units/l) 50 l

    RNaseOUT Recombinant Ribonuclease Inhibitor (40 units/l) 100 l

    E. coli RNase H (2 units/l) 50 l

    DEPC-treated water 1.2 ml

    Control RNA (50 ng/l) 15 l

    Control Primer A (10 M) 20 l

    Control Primer B (10 M) 20 l

    AdditionalMaterialsRequired

    The following items are required for use with the system, but they

    are not included:

    DNA polymerase for PCR

    Programmable thermal cycler

    Autoclaved 0.2- or 0.5-ml microcentrifuge tubes or autoclaved,thin-walled PCR tubes

    Automatic pipettes and tips capable of dispensing 120 l and

    20200 l

    Disposable gloves

    Two amplification primers specific for your target mRNA

    Microcentrifuge capable of generating a relative centrifugal

    force of 14,000 g

    37C, 42C, 65C, and 70C water baths or heat blocks

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    2

    Introduction

    SystemSummary

    The SuperScript First-Strand Synthesis System for RT-PCR is

    optimized to synthesize first-strand cDNA from purified poly(A)+ or total

    RNA. The system can be used with as little as 1 ng or as much as 5 g of

    total RNA.

    After synthesis, target cDNA can be amplified with specific primers by

    PCR without intermediate organic extractions or ethanol precipitations, as

    shown in the diagram below.

    mRNA

    AAAAAA

    (primer) TTTTTT

    First-strand synthesis

    AAAAAA

    TTTTTT

    Removal of RNA

    TTTTTT

    First-strand cDNA ready for PCR

    Continued on next page

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    3

    Introduction, Continued

    Isolation ofTotal RNA

    One important factor for synthesis of full-length cDNA is the

    isolation of intact RNA. The quality of the RNA dictates the

    maximum amount of sequence information that can be converted

    into cDNA. Thus, it is important to optimize the isolation of RNAand prevent introduction of RNases into the RNA preparation.

    RNA can be isolated by many methods. We recommend using the

    Micro-to-Midi Total RNA Purification System or TRIzol Reagent,

    which yields undegraded RNA from cultured cells or whole tissue

    samples. High-quality RNA can be purified from as little as 100

    cells up to 1 107 cells or 200 mg of tissue.

    Oligo(dT)-selection for poly(A)+ RNA is typically not necessary,

    although it may improve the yield of specific cDNAs. Poly(A)+

    RNA selection may also reduce the likelihood of genomic DNAcontamination. Frequently, RNA preparations contain small

    amounts of genomic DNA that may be amplified along with the

    target cDNA. If your application requires removal of all genomic

    DNA from your RNA preparation, refer to DNase I Digestion of

    RNA Preparation on page 18.

    First-Strand

    cDNASynthesisfrom TotalRNA

    The first-strand cDNA synthesis reaction is catalyzed by

    SuperScript

    II Reverse Transcriptase (RT). This enzyme has beenengineered to reduce the RNase H activity (found in other RTs) that

    degrades mRNA during the first-strand reaction.

    Use of an RT with reduced RNase H activity results in greater full-

    length cDNA synthesis and higher yields of first-strand cDNA than

    obtained with other RTs. SuperScript II RT has been engineered to

    retain the full DNA polymerase activity found in RNase H+ M-

    MLV RT. This further improves the enzymes ability to copy long

    RNA as compared to M-MLV RT or other derivatives. Because

    SuperScript

    II RT is not inhibited significantly by ribosomal andtransfer RNA, it may be used effectively to synthesize first- strand

    cDNA from a total RNA preparation. The enzyme exhibits

    increased thermal stability and may be used at temperatures up to

    50C.

    This system has been optimized to synthesize first-strand cDNA

    from varying amounts of starting material. The SuperScript II

    concentration has been lowered and RNaseOUT Recombinant

    RNase Inhibitor has been added to the system as part of this

    optimization process. Additionally, reaction conditions have beenmodified to further increase the sensitivity of the system.

    Continued on next page

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    5

    First-Strand cDNA Synthesis

    ProcedureOverview

    This procedure is designed to convert 1 ng to 5 g of total RNA or

    50 to 500 ng of poly (A)+ RNA into first-strand cDNA. Please

    review all the protocols before using the system.

    A Control RNA is included in the system to verify performance.

    Use 1 l (50 ng) of Control RNA as a template for first-strand

    synthesis. Refer to Troubleshooting on page 11 for additional

    information.

    Continued on next page

    RNA + Primer + dNTPS

    65C for 5 min

    Place on ice for at least 1 min

    Oligo (dT)12-18 or GSP

    Add all components except

    1 l of SuperScript

    II RT

    Mix gently

    Centrifuge briefly

    Place tubes at 42C for 2 minAdd 1 l of SuperScript

    II RT

    Mix gently

    42C for 50 min

    70C for 15 min

    Add 1 l of RNase H

    Mix gently

    37C for 20 min

    PCR or store at 20C

    Denature:

    Anneal:

    cDNA Synthesis:

    Terminate Reaction:

    Remove RNA:

    25C for 10 min

    Random Hexamers

    Add all components except

    1 l of SuperScript

    II RT

    Mix gently

    Centrifuge briefly

    Place tubes at 25C for 2 minAdd 1 l of SuperScript

    II RT

    Mix gently

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    6

    First-Strand cDNA Synthesis, Continued

    If you have >5 g of total RNA, increase reaction volumes and

    amount of SuperScript II RT proportionally.

    First-StrandSynthesisUsingOligo(dT) or aGSP

    1. Mix and briefly centrifuge each component before use.

    2. Prepare RNA/primer mixtures in sterile 0.2- or 0.5-ml tubes

    as follows:

    Component Sample No RT Control Control RNA

    up to 5 g total RNA n l n l

    Control RNA (50 ng/l) 1 l

    10 mM dNTP mix 1 l 1 l 1 l

    Oligo(dT)12-18 (0.5 g/l) 1 l 1 l 1 l

    or

    2 M GSP 1 l 1 l

    DEPC-treated water to 10 l to 10 l to 10 l

    3. Incubate each sample at 65C for 5 min, then place on ice for

    at least 1 min.

    4. Prepare the following reaction mixture, adding eachcomponent in the indicated order. (Forn samples + 1 no RT

    control + 1 Control RNA reaction,prepare the reaction mix

    forn + 3 reactions.)

    Component Each Rxn 4 Rxns

    10X RT buffer 2 l 8 l

    25 mM MgCl2 4 l 16 l

    0.1 M DTT 2 l 8 l

    RNaseOUT

    Recombinant RNase Inhibitor 1 l 4 l5. Add 9 l of reaction mixture to each RNA/primer mixture,

    mix gently, and collect by brief centrifugation.

    6. Incubate at 42C for 2 min.

    7. Add 1 l (50 units) of SuperScript II RT to each tube except

    the no RT control, mix, and incubate at 42C for 50 min.

    8. Terminate the reactions at 70C for 15 min. Chill on ice.

    9. Collect the reactions by brief centrifugation. Add 1 l ofRNase H to each tube and incubate for 20 min at 37C before

    proceeding to Amplification of the Target DNA on page 8.

    Continued on next page

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    7

    First-Strand cDNA Synthesis, Continued

    For most RT-PCR applications, 50 ng of random hexamers per 5 g

    of total RNA is adequate. Increasing hexamers to 250 ng per 5 g

    of RNA may increase yield of relatively small PCR products

    (

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    8

    Amplification of the Target cDNA

    ProcedureOverview

    The first-strand cDNA obtained in the previous procedure may be

    amplified directly using PCR. Use only 10% of the first-strand

    reaction (2 l) for PCR. Adding larger amounts of the first-strand

    reaction may actually decrease the amount of product synthesized.PCR can use several polymerases.

    Annealing and extension conditioning are primer and template

    dependent, and therefore must be determined empirically.

    PCR forTargets up to4 kb

    Use Taq DNA Polymerase or Platinum

    Taq DNA Polymerase.PlatinumTaq DNA Polymerase provides automatic hot-start

    conditions for increased specificity and sensitivity.

    1. Add the following to a 0.2- or 0.5-ml thin-walled PCR tube:

    Volume (l)Component 1 Rxn 10 Rxns

    10X PCR buffer minus Mg 5 50

    50 mM MgCl2 1.5 15

    10 mM dNTP mix 1 1010 M sense primer 1 10

    10 M antisense primer 1 10

    Taq DNA Polymerase (5 units/l) or

    PlatinumTaq DNA Polymerase (5 units/l) 0.4 4

    cDNA (from the first-strand reaction) 2 20

    autoclaved, distilled water 38.1 381

    final volume 50 500

    Note: The concentration of MgCl2 listed above is approximate. Foroptimal amplification, determine the optimal MgCl2

    concentration for your primers and template.

    2. Mix gently and overlay with silicone oil or mineral oil if the

    thermal cycler lacks a heated lid.

    3. Incubate at 94C for 2 min, then perform 2040 cycles of

    PCR with optimized conditions for your sample (1 min/kb

    extension at 6872C).

    4. Analyze 10 l of the amplified sample using agarose gelelectrophoresis.

    Continued on next page

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    Amplification of the Target cDNA, Continued

    Annealing and extension conditions are dependent on the primers

    and template, and therefore must be determined empirically.

    MaximumFidelity PCRfor Targetsup to 12 kb

    PlatinumPfx DNA Polymerase possesses a proofreading 3 to 5

    exonuclease activity and provides the highest fidelity of any DNA

    polymerase for PCR.

    1. Add the following to a 0.2- or 0.5-ml, thin-walled tube at

    either ambient temperature or on ice:

    Volume (l)Component 1 Rxn. 10 Rxns.

    10XPfx amplification buffer 5 50

    50 mM MgSO4 1 10

    10 mM dNTP mix 1.5 15

    10 M sense primer 1.5 15

    10 M antisense primer 1.5 15

    PlatinumPfx DNA Polymerase (2.5 U/l) 0.5 5

    cDNA (from the first-strand reaction) 2 20

    DEPC-treated water 37 370final volume 50 500

    Note: For most targets 1.25 units is sufficient. When amplifying

    longer targets, above 3 kb, use 2.5 units and increase

    magnesium (to 1.5 mM).

    Note: The concentration of MgSO4 listed above is approximate.

    For optimal amplification, determine the optimal MgSO4

    concentration for your primers and template.

    2. Mix gently and overlay with silicone oil or mineral oil if the

    thermal cycler lacks a heated lid.

    3. Incubate at 94C for 2 min, then perform 2040 cycles of

    PCR with optimized conditions for your sample (1 min/kb

    extension time at 6872C).

    4. Analyze 10 l of the amplified sample by agarose gel

    electrophoresis.

    Continued on next page

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    11

    Troubleshooting

    Problem Possible Cause Suggested Solution

    No bands after

    electrophoretic

    analysis of

    amplified products

    Procedural error in first-

    strand cDNA synthesis

    Use the Control RNA to verify the efficiency of

    the first-strand reaction (see the next page on

    troubleshooting with the Control RNA).

    RNase contamination Add Control RNA to sample to determine if

    RNase is present in the first-strand reaction.

    Maintain aseptic conditions to prevent RNase

    contamination.

    Use RNaseOUT Recombinant RNase Inhibitor

    in the first-strand reaction.

    Polysaccharidecoprecipitation of RNA

    Precipitate RNA with lithium chloride (seepage 19) to remove polysaccharides.

    Target mRNA contains

    strong transcriptional

    pauses

    Use random hexamers instead of oligo(dT) in

    the first-strand reaction.

    Maintain an elevated temperature after the

    annealing step (see page 16, First-strand cDNA

    Synthesis of Transcripts with High-GC

    Content).

    Increase the temperature of first-strand reaction

    (up to 50C).

    Use PCR primers closer to the 3 terminus of the

    target cDNA.

    Too much first strand

    product was used in

    PCR

    Use no more than 1/10 of the first-strand

    reaction.

    GSP was used for first-

    strand synthesis

    Try another GSP or switch to oligo(dT). Make

    sure the GSP is the antisense sequence.

    Inhibitors of RT present Remove inhibitors by ethanol precipitation of

    the mRNA preparation before the first-strand

    reaction. Include a 70% (v/v) ethanol wash of

    the mRNA pellet.

    Note: Inhibitors of RT include sodium dodecyl

    sulfate (SDS), EDTA, guanidinium salts,

    formamide, sodium pyrophosphate, and

    spermidine.

    Test for the presence of inhibitors by mixing1 g of control RNA with 1 g of sample RNA

    and comparing yields of first-strand cDNA.

    Continued on next page

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    12

    Troubleshooting, Continued

    Problem Possible Cause Suggested Solution

    Unexpected bands

    after electrophoretic

    analysis

    Contamination by

    genomic DNA

    Pretreat RNA as described in DNase I Digestion

    of RNA Preparation on page 18.

    Design primers that anneal to sequence in exons

    on both sides of an intron or exon/exon

    boundary of the mRNA to of amplified allow

    differentiation between amplification of cDNA

    and products potential contaminating genomic

    DNA.

    To test if products were derived from DNA, do

    the No RT control.

    Nonspecific annealing

    of primers

    Vary the annealing conditions. Use Platinum

    Taq DNA Polymerase for automatic hot-start

    PCR.

    Optimize magnesium concentration for each

    template and primer combination.

    Primers formed dimers Design primers without complementary

    sequences at the 3 ends.

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    13

    Troubleshooting with the Control RNA

    Introduction The Control RNA provided with this System is an 891-bp, in vitrotranscribed RNA from the chloramphenicol acetyltransferase (CAT)

    gene that has been engineered to contain a 3 poly(A) tail.

    When used with Control Primers A and B in RT-PCR, the ControlRNA will result in a 500-bp product (see page 15).

    First-strandcDNASynthesiswith Control

    RNA

    Up to 1 g of your total RNA preparation may be added to the

    following mixture to evaluate the effect of this RNA on first-strand

    synthesis and subsequent amplification.

    1. Label two autoclaved 0.5-ml tubes A and B. Tube A

    will have an addition of radioisotope to determine the

    efficiency of first-strand synthesis. An aliquot from tube B

    will be used for amplification.

    2. Prepare the RNA/primer mixtures in sterile 0.5-ml tubes as

    follows:Volume (l)

    Component Tube A Tube B

    Control RNA 1 1

    Oligo(dT) 1 1

    DEPC-treated water 7 810 mM dNTP mix 1 1

    final volume 9 10

    3. Incubate each sample at 65C for 5 min and place on ice for

    at least 1 min. Collect by brief centrifugation and add the

    following to each tube:Volume (

    l)

    Component Tube A Tube B

    10X RT buffer 2 2

    25 mM MgCl2 4 4RNaseOUT RNase Inhibitor (40 U/l) 1 1

    0.1 M DTT 2 2

    4. Incubate at 42C for 2 min.

    5. Add 1 l of [-32P]dCTP (3,000 Ci/mmol; 10 mCi/ml) to

    tube A.

    6. Add 1 l (50 units) of SuperScript II RT to each tube.

    Final volume will be 20

    l.

    7. Mix gently and collect the reaction by brief centrifugation.

    8. Incubate at 42 for 50 min (continued on next page).

    Continued on next page

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    14

    Troubleshooting with the Control RNA, Continued

    First-strandcDNASynthesiswith ControlRNA,Continued

    9. Terminate both reactions at 70C for 15 min. Place on ice

    for 1 min.

    10. Collect the reaction by brief centrifugation. Add 1 l ofRNase H to tube B and incubate for 20 min at 37C. Proceed

    with tube B to Amplification of the Control-Synthesized

    cDNA on page 15.

    11. Add 80 l of distilled water to tube A and mix gently.

    12. Remove two 5-l aliquots from tube A and spot the aliquots

    onto glass fiber filters. Dry one of the filters under a heat

    lamp or at room temperature. This filter will be used to

    determine the specific activity of the dCTP reaction.13. Wash the other filter three times, 5 min each time, with

    50 ml of ice-cold, 10% (w/v) TCA containing 1% (w/v)

    sodium pyrophosphate. Wash the filter once with 50 ml of

    95% ethanol at room temperature for 2 min. Dry the filter

    under a heat lamp or at room temperature. This filter will be

    used to determine the yield of first-strand cDNA.

    14. Count both filters in standard scintillant to determine the

    amount of32P in the reaction, as well as the amount of32P

    that was incorporated.

    15. Using equation 1, determine the specific activity (SA) of the

    dCTP in the first-strand reaction from the counts obtained

    from the unwashed filter.

    SA (cpm/pmol dCTP) = cpm/5 l

    500 pmol dCTP/5 l

    16. Using equation 2, determine the yield of cDNA from the

    counts obtained from the washed filter and the specific

    activity calculated from the unwashed filter:

    Amount of cDNA (g) = (cpm) (100 l/5 l) (4 pmol dNTP/pmol dCTP)

    (SA) (3,030 pmol dNTP/g cDNA)

    You can assume that the yield calculated for the labeled

    cDNA in tube A is equivalent to that of the unlabeled cDNA

    in tube B.

    17. Following first-strand cDNA synthesis using the Control

    RNA, you may wish to analyze the remaining labeled cDNA

    in tube A by alkaline agarose gel electrophoresis ordenaturing PAGE.

    The first-strand yield using the Control RNA should be 3050%.

    Electrophoretic analysis should yield a prominent 891-bp band

    representing 2550% of the cDNA synthesized.

    Continued on next page

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    15

    Troubleshooting with the Control RNA, Continued

    Amplificationof theControl-SynthesizedcDNA

    Following first-strand cDNA synthesis from the Control RNA,

    Control Primers A and B can be used in the following PCR

    procedure.

    Primer A (anti-sense primer): 5GAC ATG GAA GCC ATC ACA GAC3

    Primer B (sense primer): 5AGA CCG TTC AGC TGG ATA TTA C3

    Electrophoretic analysis of DNA products amplified using the

    control primers should yield a prominent 500-bp band.

    1. Perform serial dilutions with the control cDNA from tube B

    with DEPC-treated or sterile, distilled water. Dilute at

    1:1,000, 1:10,000, 1:100,000, and 1:1,000,000.

    Note: If you assume that 1% of the total RNA is mRNA, then

    50 ng of Control RNA in the first-strand reaction is

    equivalent to the amount of mRNA in 5 g of total RNA.

    One microliter of the 1:1,000,000 dilution of the first-strand

    reaction should contain ~5,000 molecules of cDNA.

    2. For each concentration, prepare a PCR mixture. Add the

    following to a 0.2-ml tube sitting on ice:

    Volume (l)Component 1 Rxn. 10 Rxns.

    DEPC-treated water 37.6 376

    10X PCR buffer minus Mg 5 50

    25 mM MgCl2 3 30

    10 mM dNTP mix 1 10

    Control primer A (10 M) 1 10

    Control primer B (10 M) 1 10

    dilution of cDNA from Control RNA 1 10

    Taq DNA Polymerase (5 units/l) 0.4 4

    final volume 50 500

    3. Mix the contents of the tube. Centrifuge briefly to collect the

    reaction components.

    4. Place reaction mixture in preheated (94C) thermal cycler.

    Do an initial denaturation step: 94C for 2 min.

    5. Perform 35 cycles of PCR:

    Denature 94C for 15 sec

    Anneal 58C for 30 sec

    Extend 68C for 1 min

    Note: If you are using a slow ramping machine (e.g., PE 480),

    please follow the manufacturers suggestions.

    6. Upon completion, maintain reactions at 4C.

    7. Analyze 10 l of the amplified sample, using agarose gel

    electrophoresis and ethidium bromide staining. A prominent

    500-bp band should be visible for all four concentrations.

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    16

    Additional Protocols

    First-strandcDNASynthesis ofTranscriptswith High-GCContent

    High-GC content mRNAs often contain stable intrinsic secondary

    structures that pose barriers to reverse transcriptase and/or primer

    annealing. Problems with RT-PCR due to secondary structure of the

    target mRNA often can be overcome by increasing the volume andtemperature of the RT reaction.

    For templates that require cDNA synthesis temperatures above

    50C, we recommend the ThermoScript RT-PCR System.

    ThermoScript RT is a genetically engineered mutant of avian

    retroviral reverse transcriptase with reduced RNase H activity that

    supports cDNA synthesis up to 70C.

    To avoid secondary RNA structure, shift the RNA/primer mix

    directly from 65C to 50C and prewarm the complete 2X reaction

    mix to 50C before adding it to the primer and RNA. Using athermal cycler simplifies the multiple temperature shifts and can

    help prevent formation of secondary structure in RNA.

    Continued on next page

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    17

    Additional Protocols, Continued

    Protocol This protocol is suitable for gene-specific or oligo(dT) primers, butnot random hexamers.

    1. Mix and briefly centrifuge each component before use.

    2. Prepare the RNA/primer mixture in a sterile 0.5-ml tube as

    follows:

    Component Sample No RT Control Control RNA

    1 to 5 g total RNA n l n l

    Control RNA (50 ng/l) 1 l

    Oligo(dT)1218 (0.5 g/l) 1 l 1 l 1 l

    or

    2 M GSP 1 l 1 l 1 l

    10 mM dNTP mix 2.5 l 2.5 l 2.5 l

    DEPC-treated water to 25 l to 25 l to 25 l

    3. Incubate each sample at 65C for 5 min and immediately

    transfer to 50C.

    4. Prepare the following reaction mixture, adding each

    component in the indicated order. Forn samples + 1 no RT

    control and 1 Control RNA reaction,prepare the reaction mix

    forn + 3 reactions.

    Component Each Reaction 4 Reactions

    DEPC-treated water 4 l 16 l10X RT buffer 5 l 20 l

    25 mM MgCl2 10 l 40 l

    0.1 M DTT 5 l 20 l

    RNaseOUT

    Recombinant RNase Inhibitor 1 l 4 l

    5. Prewarm the reaction mixture to 50C.

    6. To each sample incubating at 50C, add 25 l of prewarmed

    reaction mixture. Add 1 l (50 units) of SuperScript

    II RT

    to each tube except the no RT control, mix gently, and

    incubate at 50C for 50 min.

    7. Terminate the reactions at 70C for 15 min. Chill on ice.

    8. Collect the reactions by brief centrifugation. Add 1 l of

    RNase H to each tube and incubate for 20 min at 37C before

    proceeding to PCR (page 8).

    Frequently, problems associated with RT-PCR of GC-rich cDNA

    are related to PCR as well as first-strand synthesis. We recommend

    using PCRx Enhancer Solution, a PCR cosolvent, to facilitate

    efficient amplification of GC-rich sequences.

    Continued on next page

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    18

    Additional Protocols, Continued

    DNase IDigestion ofRNAPreparation

    If amplification products are detected from the PCR reaction in the

    absence of SuperScript II RT, it may be necessary to eliminate

    residual genomic DNA from the RNA sample. After confirming the

    efficiency of the first-strand synthesis reaction with the ControlRNA, use the following protocol to remove genomic DNA from the

    total RNA preparation. Amplification Grade DNase I has been

    extensively purified to remove trace ribonuclease activities

    commonly associated with other RNase-free enzyme preparations

    and does not require the addition of placental RNase inhibitor.

    The following procedure requires careful pipetting of all solutions

    so that the concentration of divalent metal cation (Mg2+ and Ca2+) is

    controlled. Because the DNase I must be heated to 65C to

    inactivate the enzyme, the concentration of free divalent metal ionsmust be low enough (

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    19

    Additional Protocols, Continued

    LithiumChloridePurificationof RNAPreparation

    Polysaccharides or small RNAs (tRNA and 5S RNA) that co-

    precipitate with mRNA may adversely affect first-strand cDNA

    synthesis. Highly purified mRNA can be recovered using the

    following protocol adapted from Sambrook et. al. (1).1. To the RNA sample in DEPC-treated water, add 0.1 volume

    8 M LiCl (RNase-free) and vortex. Incubate on ice for

    2 hours.

    2. Centrifuge at 13,000 g for 30 min at 4C.

    3. Remove the supernatant, being careful not to disturb the

    pellet (the pellet may be difficult to see). Dissolve the pellet

    in 200 l of DEPC-treated water by drawing the pellet in

    and out of a sterilized pipet tip.

    4. Repeat steps 1 through 3.

    5. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and

    2.0 volumes of absolute (100%) ethanol (20C). Place the

    tube at 20C for 30 min. Centrifuge at 13,000 g for 30

    min at 4C.

    6. Remove the supernatant carefully. Overlay the pellet with

    100 l of 70% ethanol (20C), and centrifuge at 13,000 gfor 10 min at 4C. Remove the supernatant, and air-dry the

    RNA pellet at room temperature.

    7. Dissolve the pellet in 10100 l of DEPC-treated water.

    8. If your starting cell or tissue sample was small (1 103 to

    1 105 cells), dissolve the pellet in 10 l of DEPC-treated

    water and use this entire amount for first-strand synthesis. If

    your starting sample was large (>1 106 cells),

    spectrophotometrically determine the concentration of thepurified RNA before proceeding to page 5, First-Strand

    cDNA Synthesis.

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    20

    References

    Berger, S.L. and Kimmel, A.R. (1987) Methods Enzymol152, 316.

    Bracete, A.M., Mertz, L.M., Fox, D.K. (1999)Focus

    21, 38.

    Chomczynski, P. (1993)Biotechniques Vol. 15, 532.

    Chomczynski, P. and Sacchi, N. (1987)Anal. Biochem. 162, 156.

    Compton, T. (1990) inPCR Protocols: A Guide to Methods and Applications (Innis,

    M., Gelfand, D., Sninsky, J., and White, T., eds.), p. 39, Academic Press, Inc.

    DAlessio, J. M., Gruber, C. E., Cain, C., and Noon, M. C. (1990)Focus 12, 47.

    Frohman, M.A., Dush, M.K, and Martin, G.R. (1988)Proc. Nat. Acad. Sci USA 85,

    8998.

    Gerard, G.F. (1994)Focus

    16, 102.

    Gerard, G.F., DAlessio, J.M., and Kotewicz, M.L. (1989)Focus 11, 66.

    Gerard, G.F., Schmidt, B.J., Kotewicz, M.L., and Campbell, J.H. (1992)Focus

    14, 91.

    Hu, A.W., D'Alessio, J.M., Gerard, G.F., and Kullman, J. (1991)Focus 13, 26.

    Lee, C.C. and Caskey, T. (1990) inPCR Protocols: A Guide to Methods and

    Applications (Innis, M., Gelfand, D., Sninsky, J., and White, T., eds.), p. 46,

    Academic Press, Inc.

    Sambrook J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory

    Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

    Simms, D., Cizdziel, P.E., and Chomczynski, P. (1993)Focus

    15, 99.

    Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997)Focus

    19, 46.

    Westfall, B., Sitaraman, K., Berninger, M., and Mertz, L.M. (1995)Focus 17, 62.

    Westfall, B., Sitaraman, K., Lee, J., Borman, J. and Rashtchian, A. (1999)Focus

    21,

    49.

    Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B.,

    Oka, M., and Imanaka, T. (1997)Appl. Environ. Microbiol. 63, 4504.

    Sitaraman, K., Darfler, M., and Westfall, B. (1999)Focus 21, 10.

    Nathan, M., Mertz, L., Fox, D. (1995)Focus 17, 78.

    Schwabe, W., Lee, J.E., Nathan, M., Xu, R.H., Sitaraman, K., Smith, M., Potter, R.J.,Rosenthal, K., Rashtchian, A., Gerard, G.F. (1998)Focus

    20, 30.

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    Related Products

    Product Size Cat. No.

    Products for RNA Isolation:

    FastTrack 2.0 mRNA Isolation Kit 6 reactions K1593-02

    Micro-FastTrack

    mRNA Isolation Kit 20 reactions K1520-02

    TRIzol

    Reagent 100 ml

    200 ml

    15596-026

    15596-018

    TRIzol

    LS Reagent 100 ml

    200 ml

    10296-010

    10296-028

    Micro-to-Midi Total RNA Purification System 50 reactions 12183-018

    Products for Analysis:

    E-Gel Pre-cast Agarose Gels

    0.8% Starter Pak1.2% Starter Pak

    2% Starter Pak

    4% Starter Pak

    9 gels and base9 gels and base

    9 gels and base

    9 gels and base

    G5000-08G5000-01

    G5000-02

    G5000-04

    UltraPure Agarose 100 g

    500 g

    15510-019

    15510-027

    UltraPure Agarose 1000 100 g 10975-035

    100-bp DNA Ladder 50 g 15628-019

    123-bp DNA Ladder 100 g

    250 g

    15613-011

    15613-029

    1-Kb Plus DNA Ladder 250 g

    1,000 g

    10787-018

    10787-026

    Enzymes for Amplification:

    Taq DNA Polymerase, Native 100 units

    500 units

    1,500 units (3 500 units)

    18038-018

    18038-042

    18038-067

    Taq DNA Polymerase, Recombinant 100 units

    500 units

    1,500 units(3 500 units)

    10342-053

    10342-020

    10342-046

    PlatinumTaq DNA Polymerase 100 reactions

    250 reactions

    500 reactions

    5000 reactions

    10966-018

    10966-026

    10966-034

    10966-083

    PlatinumTaq DNA Polymerase High

    Fidelity

    100 reactions

    500 reactions

    5,000 reactions

    11304-011

    11304-029

    11304-102

    Platinum

    Pfx DNA Polymerase(includes PCRx Enhancer Solution) 100 reactions250 reactions

    500 reactions

    11708-01311708-021

    11708-039

    Continued on next page

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    Related Products, Continued

    Product Size Cat. No.

    Enzymes for Amplification, continued:

    eLONGase Enzyme Mix 100 reactions

    500 reactions

    10480-010

    10480-028PCR SuperMix 100 reactions 10572-014

    PCR SuperMix High Fidelity 100 reactions 10790-020

    Platinum PCR SuperMix 100 reactions 11306-016

    Other Modifying Enzymes:

    DNase I, Amplification Grade 100 units 18068-015

    RNaseOUT Recombinant 5,000 units 10777-019

    Ribonuclease Inhibitor

    Ribonuclease H 30 units120 units

    18021-01418021-071

    SuperScript II Reverse Transcriptase 2,000 units

    10,000 units

    4 10,000 units

    18064-022

    18064-014

    18064-071

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    Purchaser Notification

    Limited Use Label

    License No: 4:Products for PCR

    that include norights to perform

    PCR

    This product is optimized for use in the Polymerase Chain Reaction (PCR)

    covered by patents owned by Roche Molecular Systems, Inc. and F.

    Hoffmann-La Roche, Ltd. (Roche). No license under these patents to use

    the PCR process is conveyed expressly or by implication to the purchaserby the purchase of this product. A license to use the PCR process for

    certain research and development activities accompanies the purchase of

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    Limited UseLabel License 5:SuperScript

    Reverse

    Transcriptase

    The purchase of this product conveys to the buyer the non-transferable right

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    Continued on next page

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    24

    Purchaser Notification, Continued

    Limited Use

    Label LicenseNo: 18:

    RNaseOUT

    Ribonuclease

    Inhibitor

    This product is the subject of U.S. Patent No. 5,965,399 owned by

    Invitrogen Corporation. The purchase of this product conveys to the buyer

    the non-transferable right to use the purchased amount of the product and

    components of the product in research conducted by the buyer (whether thebuyer is an academic or for-profit entity). The buyer cannot sell or otherwise

    transfer (a) this product (b) its components or (c) materials made using this

    product or its components to a third party or otherwise use this product or its

    components or materials made using this product or its components for

    Commercial Purposes. The buyer may transfer information or materials

    made through the use of this product to a scientific collaborator, provided

    that such transfer is not for any Commercial Purpose, and that such

    collaborator agrees in writing (a) to not transfer such materials to any third

    party, and (b) to use such transferred materials and/or information solely for

    research and not for Commercial Purposes. Commercial Purposes means any

    activity by a party for consideration and may include, but is not limited to:(1) use of the product or its components in manufacturing; (2) use of the

    product or its components to provide a service, information, or data; (3) use

    of the product or its components for therapeutic, diagnostic or prophylactic

    purposes; or (4) resale of the product or its components, whether or not such

    product or its components are resold for use in research. Invitrogen

    Corporation will not assert a claim against the buyer of infringement of the

    above patents based upon the manufacture, use or sale of a therapeutic,

    clinical diagnostic, vaccine or prophylactic product developed in research by

    the buyer in which this product or its components was employed, provided

    that neither this product nor any of its components was used in themanufacture of such product. If the purchaser is not willing to accept the

    limitations of this limited use statement, Invitrogen is willing to accept

    return of the product with a full refund. For information on purchasing a

    license to this product for purposes other than research, contact Licensing

    Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad,

    California 92008. Phone (760) 603-7200. Fax (760) 602-6500.

    OtherTrademarks

    TRIzol is a registered trademark of Molecular Research Center, Inc.

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