遺伝子発現解析編 - 【TOYOBO】東洋紡 ??以前検討したトランスフェリンリセプター(TFR)遺伝子の発現解析を例として事例を作成すること。(Vol.2参照) ②初心者の研究者にも分かりやすいような事例を作成すること。 ヒトTFR遺伝子は、アクセッションNO.X01060の配列を参照

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  • - TEL-- FAX--

    order_lifescience@bio.toyobo.co.jp

    - TEL-- FAX--

    order_lifescience@bio.toyobo.co.jpE-mail

    E-mail

    [http://www.toyobo.co.jp/bio] Toyobo Web Site

    TEL--

    E-mail techosk@bio.toyobo.co.jp

    FAX--

    Toyobo

    06142006.12.12,500

    PCR

    VOL.4

    8

    A TK AK T

    A

    PCRPolymerase Chain ReactionF. Hoffman-La Roche4,683,1954,683,202PCRF. Hoffman-La RochePCRSYBRMolecular Probes Inc.TaqManRoche Molecular Systems Inc.

    S M

    &QA

    PCRRT

    TaqMan ealtime PCR Master Mix 2

    SYBR Green ealtime PCR Master Mix 2

    SYBR Green ealtime PCR Master Mix -Plus- 2

    2001ml5 401ml

    2001ml5 401ml

    2001ml5 401ml

    TaqMan RNA-direct ealtime PCR Master Mix 2Mn

    RNA-direct SYBR Green ealtime PCR Master Mix 2Mn

    1000.5ml5 400.5ml2

    1000.5ml5 400.5ml2

    PCR

    Taq

    Hot start

    TA

    KOD

    PCR

    Taq

    T7

    KOD -Plus-

    KOD -Plus- Ver.2

    Blend Taq

    Blend Taq -Plus-

    TArget Clone

    TArget Clone -Plus-

    rTaq DNA Polymerase

    anti-Taq high

    ScriptMAX Thermo T7 Transcription Kit

    200U1

    200U1

    250U1

    250U1

    10

    10

    250U1

    100l

    60

    LineGene 1

    KOD-201

    KOD-211

    BTQ-101

    BTQ-201

    TAK-101

    TAK-201

    TAP-201

    TCP-101

    TSK-101

    BFFQD-33A

    RevaTra Ace -- 100

    QPK-101 QPK-101T

    QPK-201 QPK-201T

    QPK-211 QPK-211T

    QRT-101 QRT-101T

    QRT-201 QRT-201T

    FSK-101

    30,000

    32,000

    19,000

    21,000

    12,000

    16,000

    25,000

    16,000

    28,000

    1,890,000

    37,000 10,000

    37,000 10,000

    37,000 10,000

    33,000 17,000

    33,000 17,000

    53,000

    2PCRPCR ReverTra Ace --RT

    1PCRPCR PCRTth DNA polymerase

    /A/B6.021014

    Ang/l

    B

    DNA 1bp660RNA 1b330

    A

    A

    A

    A

    M

    M

    M

    M

    Ct

    Ct

    PCR

    PCR

    RNA

    TATArget Clone

    T7T3

    T7T3 RNA

    T7

    ScriptMAX Thermo T7 Transcription kit

    Code No.:TSK-101

    DNase

    DNA

    /

    /

    RNase free

    Code No.

    DNA

    53 DNA

    5 3

    5 Mn

    RNA3

    5 3

    5 3

    5 3

    TaqMan

    TaqMan

    11RT-PCR

    PCR

    53

    Taq / Tth DNA polymerase etc.

    35 Proof-readingDNA KOD DNA polymerase etc.

    DNA Tth DNA polymerase

    35

    http://www.toyobo.co.jp/bio Vol.1PCR Vol.2 Vol.3

    F: Q:

  • 1 2

    VOL.4

    PCR

    DW 10rTaq Buffer dNTPs 25mM MgCl2 F primer 10pmole/l R primer10pmole/l Taq +Anti-Taq high 1:1 Sample Total

    27l 5 5 6 2 2 1 2 50 Anti-Taq high Code No.:TCP-101Taq

    Taq DNA polymerase 5U/l1:110 -20

    55

    2 A

    PCR

    TFRVol.2

    TFRNO.X01060

    1 2 3 4 5 61 : 105 2 : 104 3 : 103 4 : 102 5 : 101 6 : 0

    40cycles

    4 15sec

    72

    10sec

    Tm-5 5sec

    94

    30sec

    94

    1 1F GATTCCTGGTTCGGGTGTTA 1R GGAAGTAACTCAACCCTAAC 40

    1-1 PCR

    PCRBacteriaTaqDNAFamily APol35ArchaeaKODDNAFamily B2Vol.1 35KOD DNA polymeraseKOD -Plus-KOD -Plus- Ver.2 TaqDNAKODDNA553PCRTaqManTaqMan Thermus thermophilusTaqTth DNA polymeraseMn2+ Biochemistry, 30: 7661-7666 1991RT-PCR11TaqTth53TaqManTth DNA polymerase3

    2-1

    ATFR mRNAPCRPCR80180bpATFRTFR DNAA4700492020merGC4555kbA200bpA1F1R

    1-2

    PCR2A3-AACTCGG-5B3-AAGTCGT-525-GAGCC-3 5-TTG-3Taq DNA polymerase3Nucleic Acids Res., 20, 4567-45731992 CT23

    ATTK2

    AK

    PCRA

    PCRTK

    A2

    2 PCR

    1 PCR AMMA 2pg10535PCRAN.C.DNAPCRM1M342A

    ARealtime PCRMPCR Taq DNA polymerase Code No.:TAP-201 TaqTaqAnti-Taq high Code No.:TCP-101AM#1AMM APCRTFR4.5KbVol.22pg

  • 1 2

    VOL.4

    PCR

    DW 10rTaq Buffer dNTPs 25mM MgCl2 F primer 10pmole/l R primer10pmole/l Taq +Anti-Taq high 1:1 Sample Total

    27l 5 5 6 2 2 1 2 50 Anti-Taq high Code No.:TCP-101Taq

    Taq DNA polymerase 5U/l1:110 -20

    55

    2 A

    PCR

    TFRVol.2

    TFRNO.X01060

    1 2 3 4 5 61 : 105 2 : 104 3 : 103 4 : 102 5 : 101 6 : 0

    40cycles

    4 15sec

    72

    10sec

    Tm-5 5sec

    94

    30sec

    94

    1 1F GATTCCTGGTTCGGGTGTTA 1R GGAAGTAACTCAACCCTAAC 40

    1-1 PCR

    PCRBacteriaTaqDNAFamily APol35ArchaeaKODDNAFamily B2Vol.1 35KOD DNA polymeraseKOD -Plus-KOD -Plus- Ver.2 TaqDNAKODDNA553PCRTaqManTaqMan Thermus thermophilusTaqTth DNA polymeraseMn2+ Biochemistry, 30: 7661-7666 1991RT-PCR11TaqTth53TaqManTth DNA polymerase3

    2-1

    ATFR mRNAPCRPCR80180bpATFRTFR DNAA4700492020merGC4555kbA200bpA1F1R

    1-2

    PCR2A3-AACTCGG-5B3-AAGTCGT-525-GAGCC-3 5-TTG-3Taq DNA polymerase3Nucleic Acids Res., 20, 4567-45731992 CT23

    ATTK2

    AK

    PCRA

    PCRTK

    A2

    2 PCR

    1 PCR AMMA 2pg10535PCRAN.C.DNAPCRM1M342A

    ARealtime PCRMPCR Taq DNA polymerase Code No.:TAP-201 TaqTaqAnti-Taq high Code No.:TCP-101AM#1AMM APCRTFR4.5KbVol.22pg

  • 3 4

    VOL.4

    5 B10nDNA

    PCR PCR

    4 A B

    M 1 2 3 4 5 6

    7 21 Total RNA-actinCtPrimer ABICheartPrimer AB SNP1SNP 6

    32 SYBR Green Realtime PCR Master MixSYBR Green Realtime PCR Master Mix -Plus-PCRDW Master Mix Primer F 10pmoles/l Primer R 10pmoles/l Total

    cDNA

    4.2-X 5 0.4 0.4 10

    X

    LineGeneBioflux

    40cycles

    15sec

    95

    15sec

    60 30sec

    Data collection

    72 60sec

    95

    22ReverTra Ace --RT

    RNase Free H2O 5RT Buffer

    dNTPs RNase inhibitor

    Random primer25pmoles/l or oligo dT primer10pmoles/l or specific primer10pmoles/l

    ReverTra Ace Total

    Poly A+ RNA 10-100ng or Total RNA 10-1000ng

    11-Xl 4 1 2 1 1 20

    X

    30min

    42 5min

    99

    4 10min

    30

    3 M 100bp Ladder marker 1 2F-3R 2 1F-3R1 3 1F-2R2 4 1F-1R4 5 1F-4R 6 1F-5RGC

    Random primer

    RT42 PCR

    RT61 PCR

    A

    B

    2331F1R21F2R11F3RM11F5R33GC34GC75%5R4R2F3R Total RNA

    2-2

    2-3 21

    APCRAPCRMM PCR3SYBR Green PCRDNA22DNASYBR Green PCRDNATm2DNA50%1DNAMelting temperature45210BBAMSYBR Green Realtime Master Mix -Plus- Code No.: QPK-211

    212RT-PCR2PCRTth DNA polymerase RT-PCR2oligodTrandomspecific primerspecifec primer PCRTth2RNA 2cDNAPCRcDNA#23

    TaqManDNADNATaqManTaqMan DNA235FRET

    2 Realtime PCR Master Mix TaqMan SYBR Green Realtime PCR Master Mix SYBR Green Realtime PCR Master Mix -Plus-

    1 RNA-direct TM Realtime PCR Master Mix TaqMan RNA-direct TM SYBR Green Realtime PCR Master Mix -Plus-

  • 3 4

    VOL.4

    5 B10nDNA

    PCR PCR

    4 A B

    M 1 2 3 4 5 6

    7 21 Total RNA-actinCtPrimer ABICheartPrimer AB SNP1SNP 6

    32 SYBR Green Realtime PCR Master MixSYBR Green Realtime PCR Master Mix -Plus-PCRDW Master Mix Primer F 10pmoles/l Primer R 10pmoles/l Total

    cDNA

    4.2-X 5 0.4 0.4 10

    X

    LineGeneBioflux

    40cycles

    15sec

    95

    15sec

    60 30sec

    Data collection

    72 60sec

    95

    22ReverTra Ace --RT

    RNase Free H2O 5RT Buffer

    dNTPs RNase inhibitor

    Random primer25pmoles/l or oligo dT primer10pmoles/l or specific primer10pmoles/l

    ReverTra Ace Total

    Poly A+ RNA 10-100ng or Total RNA 10-1000ng

    11-Xl 4 1 2 1 1 20

    X

    30min

    42 5min

    99

    4 10min

    30

    3 M 100bp Ladder marker 1 2F-3R 2 1F-3R1 3 1F-2R2 4 1F-1R4 5 1F-4R 6 1F-5RGC

    Random primer

    RT42 PCR

    RT61 PCR

    A

    B

    2331F1R21F2R11F3RM11F5R33GC34GC75%5R4R2F3R Total RNA

    2-2

    2-3 21

    APCRAPCRMM PCR3SYBR Green PCRDNA22DNASYBR Green PCRDNATm2DNA50%1DNAMelting temperature45210BBAMSYBR Green Realtime Master Mix -Plus- Code No.: QPK-211

    212RT-PCR2PCRTth DNA polymerase RT-PCR2oligodTrandomspecific primerspecifec primer PCRTth2RNA 2cDNAPCRcDNA#23

    TaqManDNADNATaqManTaqMan DNA235FRET

    2 Realtime PCR Master Mix TaqMan SYBR Green Realtime PCR Master Mix SYBR Green Realtime PCR Master Mix -Plus-

    1 RNA-direct TM Realtime PCR Master Mix TaqMan RNA-direct TM SYBR Green Realtime PCR Master Mix -Plus-

  • 5 6

    VOL.4

    11

    1

    4 RNA-direct SYBR Green Realtime PCR Master Mix PCRDW Master Mix Primer F10pmoles/l Primer R10pmoles/l RNA Total

    2.2 5 0.4 0.4 2 10

    LineGene

    12

    8

    10 LineGene -actionTFR

    45cycles

    15sec

    95

    15sec

    55 30secData collection

    74 30sec

    95

    20min

    61 30sec

    90

    9

    RNA

    RNA DNA

    LineGene

    RNALog

    2-4 2-5 PCR

    AMPCRmRNART-PCR33PCRCt: Cycle thresholdThreshold

    PCRAAKPCR A1PCRRNA-direct SYBR Green Realtime PCR Master MixAKALineGeneBioflux10l DNase Total RNAARNADNase Vol.2RT-PCRDNAA2F3RTFR-actinTotal RNATFRheartPlacentaRNA

    RNA50ng/5n2l#410ng/l210

    PCRA

    39RNAVol.2KOD -Plus- Ver.2Code No.: KOD-211TFR4.5 kbTATArget Clone -Plus-; Code No.: TAK-201TA2 RNARNACt DNARTDNA

    PCRCt105 Total RNAPolyA RNAPolyA RNArRNARNA260nm -actinG3PDHRNART-PCR 83RNAABPCRA-actinB

    1Excel3011 12HeartPlacentaMuscleTFRMuscleAVol.2Muscleactin-actinMuscleTFR

  • 5 6

    VOL.4

    11

    1

    4 RNA-direct SYBR Green Realtime PCR Master Mix PCRDW Master Mix Primer F10pmoles/l Primer R10pmoles/l RNA Total

    2.2 5 0.4 0.4 2 10

    LineGene

    12

    8

    10 LineGene -actionTFR

    45cycles

    15sec

    95

    15sec

    55 30secData collection

    74 30sec

    95

    20min

    61 30sec

    90

    9

    RNA

    RNA DNA

    LineGene

    RNALog

    2-4 2-5 PCR

    AMPCRmRNART-PCR33PCRCt: Cycle thresholdThreshold

    PCRAAKPCR A1PCRRNA-direct SYBR Green Realtime PCR Master MixAKALineGeneBioflux10l DNase Total RNAARNADNase Vol.2RT-PCRDNAA2F3RTFR-actinTotal RNATFRheartPlacentaRNA

    RNA50ng/5n2l#410ng/l210

    PCRA

    39RNAVol.2KOD -Plus- Ver.2Code No.: KOD-211TFR4.5 kbTATArget Clone -Plus-; Code No.: TAK-201TA2 RNARNACt DNARTDNA

    PCRCt105 Total RNAPolyA RNAPolyA RNArRNARNA260nm -actinG3PDHRNART-PCR 83RNAABPCRA-actinB

    1Excel3011 12HeartPlacentaMuscleTFRMuscleAVol.2Muscleactin-actinMuscleTFR

  • - TEL-- FAX--

    order_lifescience@bio.toyobo.co.jp

    - TEL-- FAX--

    order_lifescience@bio.toyobo.co.jpE-mail

    E-mail

    [http://www.toyobo.co.jp/bio] Toyobo Web Site

    TEL--

    E-mail techosk@bio.toyobo.co.jp

    FAX--

    Toyobo

    06142006.12.12,500

    PCR

    VOL.4

    8

    A TK AK T

    A

    PCRPolymerase Chain ReactionF. Hoffman-La Roche4,683,1954,683,202PCRF. Hoffman-La RochePCRSYBRMolecular Probes Inc.TaqManRoche Molecular Systems Inc.

    S M

    &QA

    PCRRT

    TaqMan ealtime PCR Master Mix 2

    SYBR Green ealtime PCR Master Mix 2

    SYBR Green ealtime PCR Master Mix -Plus- 2

    2001ml5 401ml

    2001ml5 401ml

    2001ml5 401ml

    TaqMan RNA-direct ealtime PCR Master Mix 2Mn

    RNA-direct SYBR Green ealtime PCR Master Mix 2Mn

    1000.5ml5 400.5ml2

    1000.5ml5 400.5ml2

    PCR

    Taq

    Hot start

    TA

    KOD

    PCR

    Taq

    T7

    KOD -Plus-

    KOD -Plus- Ver.2

    Blend Taq

    Blend Taq -Plus-

    TArget Clone

    TArget Clone -Plus-

    rTaq DNA Polymerase

    anti-Taq high

    ScriptMAX Thermo T7 Transcription Kit

    200U1

    200U1

    250U1

    250U1

    10

    10

    250U1

    100l

    60

    LineGene 1

    KOD-201

    KOD-211

    BTQ-101

    BTQ-201

    TAK-101

    TAK-201

    TAP-201

    TCP-101

    TSK-101

    BFFQD-33A

    RevaTra Ace -- 100

    QPK-101 QPK-101T

    QPK-201 QPK-201T

    QPK-211 QPK-211T

    QRT-101 QRT-101T

    QRT-201 QRT-201T

    FSK-101

    30,000

    32,000

    19,000

    21,000

    12,000

    16,000

    25,000

    16,000

    28,000

    1,890,000

    37,000 10,000

    37,000 10,000

    37,000 10,000

    33,000 17,000

    33,000 17,000

    53,000

    2PCRPCR ReverTra Ace --RT

    1PCRPCR PCRTth DNA polymerase

    /A/B6.021014

    Ang/l

    B

    DNA 1bp660RNA 1b330

    A

    A

    A

    A

    M

    M

    M

    M

    Ct

    Ct

    PCR

    PCR

    RNA

    TATArget Clone

    T7T3

    T7T3 RNA

    T7

    ScriptMAX Thermo T7 Transcription kit

    Code No.:TSK-101

    DNase

    DNA

    /

    /

    RNase free

    Code No.

    DNA

    53 DNA

    5 3

    5 Mn

    RNA3

    5 3

    5 3

    5 3

    TaqMan

    TaqMan

    11RT-PCR

    PCR

    53

    Taq / Tth DNA polymerase etc.

    35 Proof-readingDNA KOD DNA polymerase etc.

    DNA Tth DNA polymerase

    35

    http://www.toyobo.co.jp/bio Vol.1PCR Vol.2 Vol.3

    F: Q:

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