技術(shù)手冊――從RNA提取到熒光定量

1、 Tel: 010-*( 021-*( E-mail: people RNA 1 RNA RNA RNA RNA RNA RNA PCR RNA cDNA PCR RT PCR Real-time PCR 1 2 RNA 2.1 RNA2 Tel: 010-*(021-*(E-mail: people 3 3 RNA qPCR 4 RNA RT-PCR RNA RNA RNA(tRNA RNA 80-85 10-15 RNA(mRNA RNA(rRNA rRNA tRNA mRNA RNA RNA 1 RNA ( 1 RNA 1-5 DNA RNA rRNA 85% tRNA 10-15% m

2、RNA 1-5% RNA 1 1.1 (-20 RNA TIANGEN RNAstore -70 1.2 RNA 2 2.1 / RNA RNA pH 021-*( RNA RNA E-mail: people DNA 5 Tel: 010-*( RNA RNA TRNzol-A (DP421 TRNzol(DP405 / RNA RNAsimple RNA TRNzol-A + + RNAsimple(DP419 TRNzol RNA TRNzol-A+ TRNzol RNAsimple RNA 2.2 / - RNase TIANGEN RNA RNAprep pure RNA 6 RNA

3、 1 RNA DNA ( 2 2.1 1 TRNzol-A 2 3 + RNA RNA RNAsimple RNA 20-30 RNase 75 1-2 1-2 RNA RNase-free ddH2O RNA RNA RNase RNA RNA RNA RNA 10-30 RNA 0.6 RNA TIANGEN RNA TRNzol D 2.2 RNA pH RNase-free ddH 2O RNA ( 2 RW1 DNase RW RNA RNA pH DEPC RNA 2 DNA 7 Tel: 010-*( 021-*( E-mail: people RNA RNA 1 / RNA R

4、NA 2 / / 3 DNA/RNase DNA / DNA RNase / DNA / DNase I(RT411 DNA RNase 4 RNase RNA RNA RNA RNA 8 RNA RNA RNA Northern blot RT-PCR RNA RNA RNA RNA cDNA RNA 1 RNA RNA RNA RNA 0.025( 1cm 0.15-1.0 RNA 260 320 230 280nm 260nm OD260=1 RNA RNA RNA (ug/ml=OD260 40ug/ml 40ug/ml 1ug/ml RNA OD260 RNA RNA RNA 260

5、nm RNA 2 RNA OD260/280 OD260/280 OD260/280 OD260/280 OD260/230 TE RNA 1.9-2.1 1.8 2.2 2.0 RNA OD260/280 RNA OD260/230 RNA 3 RNA 3.1 RNA A. 1.2% 0.6g 5ml 10 43.5ml 1.5ml 37% 50ml 60 RNA 9 Tel: 010-*( 021-*( E-mail: people RNA B. RNA 5-10 55 15 2.5ul 5v/cm (20cm rRNA 28SrRNA ( 3 RNA (1000-2000rpm/min

6、3 TIANGEN TRNzol-A + RNA 5 (kb M 1 2 3 95v 80-85 28S(23S 18SrRNA 1 6 5 4 2.5 2 1.5 1 18S(16SrRNA RNA 3.2 RNA 1 700-1000ng 20 (16SrRNA RNA ( 0.5M RNase RNA( rRNA 28S rRNA ( 0.5 NaOH 4 TBE 20 1-3ul 5 V/cm RNA 80-85 18S 18S rRNA RNA 28S(23S 151 2 3 4 0.5 TIANGEN RNA( RNA RNA RNA TRNzol 0.8% rRNA 18S 28

7、S 18S 28S 18S 28S 16S 18S 23S 25S 18S 26S 16S 23S 18S 28S (kb 1.9 5.0 1.9 4.7 2.0 4.1 1.5 1.9 2.9 3.7 2.0 3.8 1.5 2.9 4.0 1.8 rRNA 10 RNA DNA RNA RNA 2 3 RNase RNA RNase RNA RNA RNase RNA RNA 1 1.1 RNA RNA RNA RNA RNA RNA -80 1.2 RNA RNase RNA RNase RNase RNase RNA 1.3 RNA RNase RNA RNase 10 RNase D

8、EPC 0.1%(v/v TRNzol RNase RNase RNase 150 4 RNase RNase ( 0.5M NaOH 2 2.1 RNA RNA RNase RNA RNase 11 Tel: 010-*( 021-*( E-mail: people RNA 2.2 RNAprep pure RNase RNA TRNzol-A+ TRNzol RNAsimple RNase RNA 2.3 RNA RNase RNA RNA 2.4 RNA TIANGEN RNase Tris MOPS RNAsafe RNA RNA RNAsafe RNase RNA 1/20 RNas

9、e (60 10-20 RNase RNAsafe 60 DEPC 20 RNA RNA RNAsafe(DP409 3 RNA RT-PCR RNasin 51000kDa RNase RNA RNA Northern blot RNasin RNase pH 4.7 RNasin RNase RNase RNA 12 RT-PCR RT-PCR DNA RNA RT RNA RT-PCR cDNA cDNA cDNA cDNA RNA PCR RT-PCR 1 RNA RNA RNA DNA RNA mRNA DNA RNA DNase I RNA RNA DNA RNA TRNzol R

10、NAprep pure 2 Oligo dT mRNA Oligo dT mRNA RNA ( RNA RNAstructure PCR PCR RT (GSP RT-PCR PCR RT-PCR RT PCR RNA RT Poly A RNA DNA mRNA Oligo dT DNA RNA RNA (GSP 3 3.1 Quant Reverse Transcriptase Quant AMV M-MLV GC ( 1 RNA RNA RNA 13 Tel: 010-*( 021-*( E-mail: people RT-PCR 3.2 M-MLV TIANScript( RNA DN

11、A RNA cDNA ( 2 31 ng RNA M-MLV 71kDa DNA RNA DNA RNaseH cDNA 1000 500 250 125 62 Quant Reverse Transcriptase MMLV RNase HMMLV AMV RNase HAMV M 1 2 3 4 2 1 Quant Reverse Transcriptase AMV,M-MLV 1ug RNA -actin 1ul M:TIANGEN Marker 1 ,Oligo(dT15 2 ,Oligo(dT15 3 M-MLV,Random 4 M-MLV,Oligo(dT15 14 RT-PCR

12、 1 PCR cDNA 0.01pg RNA RT-PCR cDNA ( 3 cDNA 3 RT-PCR 2 PCR PCR RNA oligo(dT mRNA ( 4 cDNA cDNA cDNA 4 RT-PCR 3 PCR 15 Tel: 010-*( 021-*( E-mail: people RT-PCR RT-PCR RNA RNA RNA RNA PCR RNase RNA 50 ng RNA cDNA 1 mM 3 Tm 3 mM 0.1ug 0.5 mM 0.5ug BSA RNasin(DP418 RNA DNA RNA DNA DMSO SDS TRNzol RNAsto

13、re RNAprep pure RT-PCR 1 RNA cDNA EDTA RNase SDS RNA poly(A+ RNA ( RNA RNA RNA 3-5 RNase ( RNA DTT RNA RNase RNase RNase 10,000 g 5 cDNA RNase A B C RNase cDNA RNA RNA RNase 4kb RNA RNA NaCl RNA 0.2M 4 RNA RNase -70 RNA RNA RNA cDNA RNase RNA 2 RNaseH RNA RNaseH (RNaseH - cDNA RNaseH M-MLV AMV RNA D

14、NA 16 RNA cDNA RNaseH RNaseH RT RNA cDNA RNaseH 3 DMSO RNA 20 10 0.4 45 1/10 PCR 20 10 DMSO M-MLV RT-PCR PCR AMV 17 Tel: 010-*( 021-*( E-mail: people RT-PCR RT-PCR 1 -actin R actin f R actin r R actin f R actin r h actin f h actin r h actin f h actin r m actin f m actin r m actin f m actin r tcaggtc

15、atcactatcggcaat aaagaaagggtgtaaaacgca cccatctatgagggttacgc tttaatgtcacgcacgatttc tgacgtggacatccgcaaag ctggaaggtggacagcgagg cgggaaatcgtgcgtgac tggaaggtggacagcgagg gagaccttcaacaccccagc atgtcacgcacgatttccc gagaccttcaacaccccgc ccacaggattccatacccaa 432bp 150bp 205bp 434bp 263bp 446bp 2 Tm Human actin bet

16、a Rat actin beta Mouse actin beta human GAPDH Rat GAPDH Mouse GAPDH Rat 18s rRNA Human 18s rRNA Mouse 18s rRNA F305 R868 F18 R224 F91 R607 F369 R626 F50 R664 F306 R835 F1865 R2164 F1565 R1816 F56 R296 ctgggacgacataggagaaaa aaggaaggctggaagagtgc cacccgcgagtacaaccttc cccatacccaccatcacacc atatcgctgcgctg

17、gtcgtc aggatggcgtgagggagagc agaaggctggggctcatttg aggggccatccacagtcttc cagtgccagcctcgtctcat aggggccatccacagtcttc aggccggtgctgagtatgtc tgcctgcttcaccaccttct tgcggaaggatcattaacgga agtaggagaggagcgagcgacc cagccacccgagattgagca tagtagcgacgggcggtgtg aggggagagcgggtaagaga ggacaggactaggcggaaca 305-324 868-849 1

18、8-37 224-205 91-110 607-588 369-388 626-607 50-69 644-625 306-325 835-816 1865-1886 2164-2143 1565-1584 1816-1997 56-75 296-277 52.3 52.6 54.5 54.4 57.5 57.8 55.6 55.1 55 55.1 54 54.6 58.6 59 58.3 58.3 54.4 54 59.4 60.4 60.4 57.5 58.3 58.7 59.3 59 62 564bp 207bp 517bp 266bp 595bp 530bp 300bp 253bp 2

19、41bp 18 PCR PCR PCR (Real-time PCR PCR Real-time PCR (1 (2 real-time PCR PCR PCR 1996 PCR Applied Biosystems PCR PCR 90 Taq DNA PCR 5 -3 PCR PCR PCR PCR 1 Taq dNTP PCR Taq PCR PCR DNA PCR PCR DNA PCR DNA 1 96 PCR S PCR DNA ( PCR PCR A B C D : 2 96 (Taqman 19 Tel: 010-*( 021-*( E-mail: people PCR PCR

20、 1 Ct 2 PCR DNA DNA PCR PCR 2 PCR ( PCR PCR Buffer 1 n X X0 n 2n Xn=X0 2n PCR PCR PCR Xn=X0 (1+Exn 2 1 1 Ex Ct XCt=X0 (1+ExCt =M 3 XCt M (3 logM=logX0 (1+ExCt log X0= - Ct log(1+Ex + log M M Ex Ex Ex PCR 4 5 20 Log Ct 2 2.1 PCR PCR PCR PCR PCR 3 PCR PCR ( 3 Ct ( 4 4 PCR PCR PCR PCR DNA 2.2 ( 5 PCR D

21、NA 5 Ct 1250-20000 21 Tel: 010-*( 021-*( E-mail: people PCR 2.3 PCR (threshold (baseline 3 PCR 15 15 10 Ct 2.4 Ct Ct Ct Ct Ct 2.5 PCR (R E E=10-1/ PCR 100 90-105 100 90 105 10-1/ -1 =2 PCR 1 -3.32 (R2 PCR 1 1.1 SYBR Green SYBR Green ( 6 1000 DNA PCR Green 497nm SYBR Green PCR Real-time PCR (94 (50-6

22、0 6 SYBY Green DNA PCR( SYBR Green PCR SYBR Green DNA DNA DNA DNA 7 SYBR Green SYBR 520nm 22 2 (68-72 40-45 SYBR Green SYBR Green DNA PCR SYBR Green 7 SYBR Green SYBR Green 1.2 PCR DNA DNA DNA 8 (Tm DNA 50 DNA 9 PCR Tm ( 8 60 95 DNA ( 9 SNPs 2 2.1 Taqman Taqman PCR Taqman Beacon FRET PCR Taqman Taqm

23、an 23 Tel: 010-*( 021-*( E-mail: people PCR PCR DNA PCR ( TET, VIC, HEX 10 FAM, Taq 5 -3 2.2 Beacon (Beacon 15-30 5-7 10 TaqMan ( 11 FAM Texas (50- Red PCR 60 (68-72 (94 40-45 11 2.3 FRET FRET 5 3 FAM Red640 Red 640 FAM Red640 Red640 640nm 12 FRET FRET FAM 24 640nm FRET LC-Red640 LC-Red705 3 PCR SYB

24、R Green FRET +2 1 400bp GC 20 -80 GC 100-150bp 4 G 2 100200bp PCR 3 3 bp 5 bp 4 5 3 5 5 25 Tel: 010-*( 021-*( E-mail: people PCR 18-25bp GC 4 5 4 2 G G / oligo 2 ( C 30-80% Tm 3 58-60 T C G A 3 TM F(forword primer preiemer 4bp Tm R(reverse Tm DNA 3 100-200bp PCR 50bp-150bp 150bp real-time 5 ( 5 20-3

25、0bp 5-10 5 GC 40%-70% G( Blast 25pmol/ul 25pmol/ul ul (OD 33/ 5 G C Tm 3 3 65-70 G -20 40000 26 PCR PCR DNA RNA PCR SNP PCR 1 RNAi PCR PCR PCR 2 PCR cDNA PCR 3 SNP GC Methylight DNA DNA SNP SNP PCR SNPs 5 Taqman 90% SNP 27 Tel: 010-*( 021-*( E-mail: people RNA RNA RT-PCR RNA RNA RNA DNA RNA RNA 1 RN

26、Aprep pure 1.1 RNA - DP431 DP432 DP430 DP433 DP420 RNAprep pure RNA RNAprep pure RNA RNAprep pure / RNAprep pure RNA RNAprep pure RNA RNA RL RW1 RNase-free RNase-free K/ CR* (DP431 CS DP433 RNase-free ddH 2O RW RNase-free DNase I (1500u RNase-free (DP432 DP430 (DP433 10 Carrier RNA(DP420 * CR DP433

27、CR2 DP420 CR1 CR3 1 RL RL 4 1% 1 ml RL RL 10 ul - 2 3 RW 28 1.2 1 10-30mg ( 300ul RL 4 12,000 rpm(13,400 1 109 2 20-25 100 ul TE TE G G + ( 400 ul/ ml 3 mg/ ml 3-5 min 5-10 min 1 TIANamp/RNAprep pure ( ( 10cm 1 2 0.10-0.25% PBS 1 10 7 1 107 ( 300 g 5 ( PBS RNase-free 300 g PBS 5 RNA RNA 29 Tel: 010-

28、*( 021-*( E-mail: people RNA 1 5 1 ( 1 RL 3/4 10-15 2 20 4 2,100rpm (400 g 10 1 2 ( 1 1 0.1g RNase RNase RNA 590ul RNase-free ddH2O 12,000 rpm(13,400 g 2-5 10ul K 56 10-20 20mg RNA 350 ul 2 RL 12000 rpm(13,400 g RL 30 RL (ul 510 510 6-1107 RL 6 350 600 cm 6 6-10 RL (ul 350 600 RL RL(ul 350 600 0.5 0

29、.5-1.5 2 (ml 2 106 10 6 1 10 7 50-100mg 1: 56 1-3 450ul RL 2: RNA 0.5 CR 70 CS 2 1 g 30-60 70% ( CS 350ul 600ul 12,000 rpm(13,400 g CR CR CR 12,000rpm(13,400 70% RNase-free ddH 2 O CR 021-*( E-mail: people 70% 31 Tel: 010-*( RNA 12,000rpm(13,400 g 30-60 DNase I -20 DNase I 9 1500 U 550ul RNase-free

30、ddH2O -20 DNase I 4 6 DNase I CR CR CR 350 ul 80 ul 350 ul 10 ul DNase I RW1 DNase I RW1 70 ul RDD 12,000 rpm(13,400 15 12,000 rpm(13,400 g 30-60 g 30-60 CR 12,000 rpm(13,400 500 ul g 30-60 RW( CR 2 12,000 rpm(13,400 g 2 CR CR RT CR RNase-free ddH2O RNase-free 2 12,000rpm(13,400 g 2 30-100ul RNA 30u

31、l RNA -70 32 2 TRNzol/TRNzol-A+/RNAsimple 2.1 RNA 75 (DEPC DEPC Eppendorf RNase free ddH2o 0.05% DP405 DP421 TRNzol TRNzol-A+ RNA RNA TRNzol TRNzol-A+ RZ* RW RNase-free RNase-free CR3 RNase-free ddH 2O RD RNase-free DP419 RNAsimple RNA * RZ TRNzol-A+ 1 2 3 2.2 2 / RNA 1cm3 30-100mg TRNzol-A+(DP405/D

32、P421 TRNzol/TRNzol-A+ 10 30-100mg 1ml TRNzol/ / 2 TRNzol/TRNzol-A +/RNAsimple 33 Tel: 010-*( 021-*( E-mail: people RNA 1 RNA 2 RNA 1ml TRNzol/TRNzol-A (DP405/DP421 1 RNA mRNA mRNA + 1cm3 2 RNase 3 RNA 1cm3 (DP408 30-100mg 1 TIANGEN RNAstore 30-100mg 1ml TRNzol/TRNzol-A (DP405/DP421 TRNzol/TRNzol-A+(

33、DP405/DP421 + RNA 1ml RNA 3 TRNzol TRNzol TRNzol-A +( 0.25ml 0.75ml RNA 1 TRNzol/TRNzol-A+ 2 5 A + 10cm2 1ml 106-107 mRNA 1ml TRNzol/TRNzol-A+ TRNzol/TRNzol- 34 50 u l TRNzol-A+ TRNzol/TRNzol-A + TRNzol/ RNA RNA 1 1 (litycase 1M 0.1M EDTA, pH 7.4 0.1% 2 100ul 5 -mercaptoethanol 107cells 107 (litycas

34、e 4 30 2 RNA 50U lyticase/1 1000rpm 10-30 5 300rpm + 1mlTRNzol/TRNzol-A lyticase Lyticase 2 1 2 1000rpm TRNzol-A+ Eppendorf 0.45-0.55mm 1.5ml Eppendorf 5 300ul 12000rpm 2 5 10 7 1 DEPC 4 500ul TRNzol/ TRNzol/TRNzol-A+ TRNzol/TRNzol-A + TRNzol/TRNzol-A + 15-30 5 0.1g 1ml TRNzol/ TRNzol-A + 35 Tel: 01

35、0-*( 021-*( E-mail: people RNA 12000rpm DNA 5 RNA RNase 4 12,000 rpm 10 0.2 ml 15 RNase 3 1ml TRNzol/TRNzol-A+ 2 4 12,000rpm RNA 10-15 600ul DNA : 1 TRNzol TRNzol-A + 20-30 10 4 12,000rpm RNA 1 ml 75% 75% 1ml TRNzol/TRNzol-A+ 5 1ml 75% 75% DEPC DEPC RNase-free ddH2O RNA 36 2 RNAsimple RNA 0.5 CR3 CR

36、3 4 12,000 rpm (13,400 CR3 g 4 30 12,000 rpm (13,400 g 30 1 2 4 3 g 4 CR3 000 rpm (13,400 CR3 CR3 30 2ml 0.5 ml g 0.7 ml g 0.5 ml 4 30 RD RW 30 RW 2 4 g 4 2 12, 12,000 rpm (13,400 12,000 rpm (13,400 2 12,000rpm (13,400 CR RT CR3 4 12,000 rpm (13,400 g 30ul 2 30-100ul RNase-free ddH2O RNA -70 2 RNA 3

37、7 Tel: 010-*( 021-*( E-mail: people RNA RNA Q-1 RNA A1 A2 RNA A3 -70 RNA -70 ( A4 A5 RNA Q-2 OD 260 /OD 280 A1 A2 A3 A4 OD260 OD280 OD260 0.1-0.5 Q-3 OD 260 /OD 230 A1 A2 A3 OD260 OD230 OD260 0.1-0.5 RNA 75% LiCl Q-4 RNA A1 2ug/mg A2 RNA RNA RNA (2-4ug/mg (0.05ug/mg RNA (0.05- 38 RT-PCR RT PCR RNA R

38、NaseH Quant DNA RT-PCR 1 PCR 10ng-5ug total RNA ER103 KR103 ER104 KR104 KR113 Quant Reverse Transcriptase Quant cDNA TIANScript M-MLV TIANScript cDNA Quant RT-PCR Quant RT 10 RT Buffe TIANScript M-MLV First-Strand Buffe RNase-free ddH2O oligo(dT15 Random dNTP Quant RTase (for one step 10 RT-PCR Buff

39、er 5 RT-PCR enhancer RNasin dNTP Mixture RNase-free ddH2O Hotmaster Taq polymerase 1 cDNA DEPC 2 RNA 3 4 5 cDNA -20 RNA DNA -20 39 Tel: 010-*( 021-*( E-mail: people RT-PCR 2 2.1 2.1.1 RT-PCR RT Quant cDNA 1 RNA (15-25 10 5s RT mix RNasin DTT dNTP RNase-free ddH2O 2 3 4 5 37 RNA 60 5s 1 10 RT mix 2.5

40、mM each Random 10uM * 2 ul 2 ul 2 ul 1 ul X ul 4 X ul 20 ul 1 ul 1 ul 1 ul 0.5 ul X ul X ul 10 ul 1 0.25mM each dNTP 1uM 20ul dNTP Oligo-dT15 RNase-free RNA Quant Reverse Transcriptase TIANscript M-MLV 1 1-5 ug RNA 50-500ng mRNA 2ul Random 2 pmole 2 ul oligo (dT15 2 ul dNTP 2 70 4 ul 5 5 First-Stran

41、d Buffer 2.5 mM each 13.5ul 2 RNase-free ddH2O 1 ul 0.1 M DTT 0.5 ul RNasin 3 4 42 5 95 1 ul(200UTIANScript M-MLV 25 10 50 5 RNase H 6 7 40 6 7 RNase H 1 ul (2U 37 RNase-free ddH2O 20 50ul RNA 2-5ul 95 PCR 5 2.1.2 RT PCR 20ul PCR 2.1.3 2 5v/cm 15-20min 6ul 6ul MarkerI ( 2 Taq PCRMaster Mix 10 ul 0.5

42、-1ul(10uM 0.5-1ul(10uM cDNA ddH2O 2 ul 6-7 ul PCR 95 94 55 72 72 2min 15sec 15sec 15sec 2min 30 2 Quant RT jun (252bp Marker I Taq PCR MasterMix PCR 2 2 RT-PCR Quant RT-PCR 50ul GC 0.5uM oligo dT 1 2 Quant RTase 3 RT-PCR RNasin Hotmaster Taq -20 dNTP GC 41 Tel: 010-*( 021-*( E-mail: people RT-PCR 4

43、5 5 6 RNA RT-PCR enhancer dNTP mix 10 PCR 50 RT-PCR Buffer RNase-free H2O / 10 RT Buffer 5 ul 2 ul 10 ul 0.5 ul 2.5 ul 0.5 ul 3 ul 3 ul 10ng-1ug total RNA 50ul 50ul dNTP Mixture(10mM each 5 RT-PCR enhancer RNasin (40U/ul Hotmaster Taq polymerase(2.5U/ul Quant RTase(for one step (10uM (10uM RNA RNase

44、-free H2O RT-PCR 7 8 PCR PCR 50 PCR Human RNA 1 2 3 4 5 6 7 3-5 30-40 30 min 65 PCR 30 min 2 min 0.5-1 min 0.5-1 min 0.5-2 min 50 94 94 50-60 65 M HS1 HS2 HS3 HS4 HS5 HS6 HS7 3 Quant One Step RT-PCR Kit(KR113 RNA RT-PCR 9 2 5v/cm 6ul MarkerI 15-20min ( 3 6ul HS3 HS7 HS1 M TIANGEN 50bp DNA Ladder HS6

45、 GC HS2 HS4 (GC HS5 60 42 RT Q-1 A1 RT RNase RNase A2 0.8mM DTT RNA PCR RT-PCR RNA RNA 45 pH RNase SDS EDTA RNA 70 A3 25 GSP RNA 0.1-0.5ug A4 A5 RT-PCR PCR cDNA 1/5 GSP RNA BSA oligo(dT 50ng RNA 10 GSP cDNA A4 v/v RNA RNA 8.0 DTT 100 RNA Q-2 A1 3 oligo dT Taq DNA A2 A3 DNA A4 A5 A6 UDG 3 DNase I RNA PCR