AS　LMD　Sampling　Manual

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1 AS LMD Application Solutions LaserMicrodissection System LMD SamplingManual

2 AS LMD Sampling Manual DNA RNA RNA cdna PCR RNA RNA RT-PCR PCR

3 AS LMD (90FOIL-SL25) (90COVER-GLASS25) SIGMAP8920 BSA BSA BSA 25 UV RNase 2

4 AS LMD AS LMD HE RNA OTC OCT OCT AS LMD UV (337.1nm) UV UV AS LMD UV 3

5 OCT n- RNaseFree DEPC RNaseFree RNaseFree RNA OCT n- p25 OCT OCT OCT OCT OCT 15 1 RNaseFree OCT 4

6 0.05% 30sec1min 1 RNaseFree 1 RNaseFree AS LMD 5

7 96% 70% PBS RNA RNA HE AS LMD 6

8 HE PBS 5 1 7

9 Living cells AS LMDLCC Living Cell Cutting LCC AS LMD LCC AS LMD AS LMD LCC AS LMD AS LMD 8

10 Cytospins 50 1ml 1 Blood smear 1 Chromosome preparation 100x 72 / (3+1, v/v) 9

11 DNA DNA DNA DNA PCR PCR-SSCP DNA DNA 10mM Tris-HCl ph8.0 ; 1% Tween-20 ; 5mg/ml Proteinase K Proteinase K 5mg/ml 100l 100mg/ml Proteinase K 5l 20100l 55 1 over night Proteinase K Proteinase K PCR DNA DNA 1 PCR 1 PCR 2 PCR nested PCR DNA 55 DNA DNA DNA QIAGEN DNA blood/tissue kits 10

12 RNA RNA RNA RNA cdna RT-PCR RNA RNA DEPC RNA QIAGEN RNeasy Kit RNA RNA AGPC RNA QIAGEN RNAeasy Mini Kit (Cat. No ) RLT RNA GTC2-2-ME 20100l QIAGEN Rneasy RNA RNA RNA

13 RNA cdna RNA RNA DEPC cdna cdna (dt) cdna 4 µl dntp-mix; 10 mm each (Applied Biosystems N ) 4 µl MgCl2; 25mM 2 µl GeneAmp 10x Buffer II (Applied Biosystems N ) 1 µl Random hexamer primers (Applied Biosystems N ) 1 µl MuLV reverse transcriptase (Applied Biosystems N ) 1 µl RNase Inhibitor (optional) (Applied Biosystems N ) RNA DEPC PCR 12

14 PCR (Polymerase Chain Reaction) PCR PCR Tm PCR PCR 1x Taq-buffer complete with 1.5 mm MgCl2 dntp-mix (à 200µM) (Taq)-polymerase (1u) Primer 1 (2 µm) Primer 2 (2 µm) Template DNA 50l Cycle Denaturing Annealing polymerisation 1 95 C, 2 min C, 30sec. 50 C, 45sec. 72 C, 1min 1 72 C, 5min DNA 13

15 Protein-Analysis SDS-PAGE DNARNA SDS-PAGE SDS-PAGE 10% (v/v), 5% (v/v) 2-, 2% (w/v) SDS, 0,05% (w/v) BPBBromphenolblue 2 mm Tris-base M urea2% NP405% 2-14

16 RNA AS LMD RNA RNA mrna RNA mrna 1.5kb RNA RT-PCR 70% 100% 3 LMD << >> O.C.T. compound << >> 180,8h Poly-L-lysine O.C.T. Compound 20m 3 100% EtOH or 70%EtOH or EtOH/Acetic Acid 19:1 DEPC 1min 0.05%,10sec DEPC 1min2times 30sec 80 AS LMD 30min RNA 15

17 Agilent Bioanalyzer 28S18S rrna 18S rrna 28S rrna 100%EtOH 70%EtOH EtOH/Acetic Acid rrna Ratio [28S 18S] 0.66 ND RNA RNA RT-PCR PCR GAPDH CYP1A RNA Poly-A 0.3kb1.5kb PCR RT-PCR RNA mrna M 1 M CYP1A1 GAPDH 16

18 RNA RT-PCR Rnase free -80 OCT OCT m % RNA DEPC OCT DEPC % AS LMD 0.5mlPCR TRIzol 30l Bar=100m RNA PCR Spin Down ~ TRIzol 20l Spin Down ~ TRIzol 150l 2~3 40l ,000xg 15 50l A 4 12,000xg 15 A B M Ethachinmate l 75% 4 12,000xg 5 DEPC 30l 20l 17

19 RNA 4RT-PCR RNA RT-PCR PT-PCR SuperScript One-Step RT-PCR with PLATINUM Taq (LIFE TECHNOLOGIES ) /tube X Reaction Mix 25l Template RNA 6l Sense/Anti-Sense Primer(GAPDH or VEGF, 10M) l RT/PLATINUM Taq Mix 1l DEPC water to 50l ) RT-PCR Total RNA RT-PCR GAPDH VEGF 308bp 308bp 311bp 239bp GAPDH no RT/PLATINUM Taq Mix GAPDH VEGF188 (311bp) VEGF164 (239bp) 18

20 PCR autoclaving tape 200 hr 200ml OCT compound m 1 1 RNase-Free MQ wash5 0.03% 5 DEPC-MQ wash 2 70% 1 OK DEPC-MQ Cool

21 RNA RNA QIAGEN RNeasy Mini Kit RLT buffer -mercaptoethanol 10l -MERLT buffer 1ml 350l RLT buffer RPE buffer EtOH RNasefree water 70% EtOH 2025 LMD volume RNase-free water 40l RLT buffer RLT buffer lysate 350l 70%Etoh 350l RNeasy spin column 2ml-collection tube 700l lysate 10,000rpm. 15sec. r/t RWI buffer 700l RWI buffer 10,000rpm. 15sec. r/t RNeasy 2ml-collection tube 500l RPE buffer 10,000rpm. 15sec. r/t 500l RPE buffer 10,000rpm. 2min. r/t 15,000rpm. 2min. r/t RNeasy 1.5ml-collection tube 50l RNase-Free Water 1min. 12,000rpm. 1min. r/t RNA RNA RNA 10M NH4OH EtOH over night 10.8l RNase-Free Water 20

22 PCR ChAT(choline acetyltransferase) In situ hybridyzation. Bar=450m ChAT GAPDH SOD1 PCRApplied Biosystems Sequence Detection System 0 mrna easymini GAPDH C57BL/6J GluR2 (GluR2) 21

23 Kolble K. The LEICA microdissection system: design and applications. J Mol Med. 2000;78(7):B24-5. Mori M, Mimori K, Yoshikawa Y, Shibuta K, Utsunomiya T, Sadanaga N, Tanaka F, Matsuyama A, Inoue H, Sugimachi K. Analysis of the gene-expression profile regarding the progression of human gastric carcinoma. Surgery Jan;131(1 Suppl):S Inoue K, Sakurada Y, Murakami M, Shirota M, Shirota K. Detection of gene expression of vascular endothelial growth factor and flk-1 in the renal glomeruli of the normal rat kidney using the laser microdissection system. Virchows Arch Feb;442(2):

24 40%70% 70%,40% 23

25 40% 40% 24

26 25

27 SP 10,000/SEC n- -79 n- n- n- n- n

28 SP bp. Boiling point mp. Melting point bp. mp. () Water (Helium) (Nitrogen) (Oxygen) (Ethane) (Propane) (Isopentane) (Freon 12)* (Acetone) n- (n-hexane) * K/SEC 10-4K/SEC (1,000,000/SEC) (10,000/SEC) CE* ETHANE LN2 at RT*(BILING) LN2 COOLING to FP*(SLUSH) ISOPENTAN FREON * CE (LN2) (Ethan) * RT Room Temperature * FP Freezing Point 27

29 SP dyne/cm (o,m,p) 30.0, 28.6, 28.3 n (MKE) 24.6 n (MIBK)

AllPrep DNA/RNA Micro プロトコールとトラブルシューティング ( /2008)

AllPrep DNA/RNA Micro プロトコールとトラブルシューティング ( /2008) July 2007 AllPrep DNA/RNA Micro DNA RNA 5 x 10 5 5 mg Sample & Assay Technologies DNA RNA 3 DNA RNA 11 DNA RNA 19 24 2 AllPrep DNA/RNA Micro 07/2007 DNA RNA RNA AllPrep DNA Spin Column DNA RNeasy MinElute