AS LMD Application Solutions LaserMicrodissection System LMD SamplingManual 2005 1
AS LMD Sampling Manual -------------------------------------------------------------2 -------------------------------------- 2 ----------------------------------------------------- 2 ---------------------------------------------------------------------- ------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------ --------------------------------------------------------------------- ------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------ --------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------- ---------------------------------------------------------------10 DNA ------------------------------------------------------------------------------------ 10 RNA -------------------------------------------------------------------------------------11 RNA cdna ---------------------------------------------------------------------12 PCR-----------------------------------------------------------------------------------------------13 -----------------------------------------------------------------------------14 ------------------------------------------------------------------15 RNA ------------------------------15 RNA RT-PCR------------------------------------------------17 PCR----------------------------------------19 ---------------------------------------------------------------------------------------22 -----------------------------------------------------------------------------------------22 -----------------------------------------------------23 -------------26 -------------27 ----------------------------------28 1
AS LMD (90FOIL-SL25) (90COVER-GLASS25) SIGMAP8920 BSA BSA BSA 25 UV 180 8 200 2 RNase 2
AS LMD AS LMD HE RNA OTC OCT OCT AS LMD UV (337.1nm) UV UV AS LMD UV 3
OCT n- RNaseFree DEPC RNaseFree RNaseFree RNA OCT n- p25 OCT OCT OCT OCT OCT 15 1 RNaseFree OCT 4
0.05% 30sec1min 1 RNaseFree 1 RNaseFree 5 30 2030 AS LMD 5
96% 70% PBS RNA RNA 5 40 40 2 1 HE AS LMD 6
30 3 30 30 HE PBS 5 1 7
Living cells AS LMDLCC Living Cell Cutting LCC AS LMD LCC AS LMD AS LMD LCC AS LMD AS LMD 8
Cytospins 50 1ml 1 Blood smear 1 Chromosome preparation 100x 72 / (3+1, v/v) 9
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 15 99 10 Proteinase K PCR DNA DNA 1 PCR 1 PCR 2 PCR nested PCR DNA 55 DNA DNA DNA QIAGEN DNA blood/tissue kits 10
RNA RNA RNA RNA cdna RT-PCR RNA RNA DEPC RNA QIAGEN RNeasy Kit RNA RNA AGPC RNA QIAGEN RNAeasy Mini Kit (Cat. No. 74 106) RLT RNA GTC2-2-ME 20100l 15 23 QIAGEN Rneasy RNA RNA RNA -80 11
RNA cdna RNA RNA DEPC cdna cdna (dt) cdna 4 µl dntp-mix; 10 mm each (Applied Biosystems N8080260) 4 µl MgCl2; 25mM 2 µl GeneAmp 10x Buffer II (Applied Biosystems N8080010) 1 µl Random hexamer primers (Applied Biosystems N8080127) 1 µl MuLV reverse transcriptase (Applied Biosystems N8080018) 1 µl RNase Inhibitor (optional) (Applied Biosystems N8080119) RNA DEPC 20 10 42 60 PCR 12
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. 30 94 C, 30sec. 50 C, 45sec. 72 C, 1min 1 72 C, 5min DNA 13
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 10 9.5 M urea2% NP405% 2-14
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
Agilent Bioanalyzer 28S18S rrna 18S rrna 28S rrna 100%EtOH 70%EtOH EtOH/Acetic Acid rrna Ratio [28S 18S] 0.66 ND 0.18 0.35 RNA RNA RT-PCR PCR GAPDH CYP1A RNA Poly-A 0.3kb1.5kb PCR RT-PCR RNA mrna M 1 M CYP1A1 GAPDH 16
RNA RT-PCR ----- Rnase free -80 OCT ----- OCT m ----- 70% RNA DEPC 1 -----OCT DEPC -----0.05% 30 ----- AS LMD 0.5mlPCR TRIzol 30l Bar=100m RNA PCR Spin Down ~ TRIzol 20l Spin Down ~ TRIzol 150l 2~3 40l 3 4 12,000xg 15 50l A 4 12,000xg 15 A B M Ethachinmate l 75% 4 12,000xg 5 DEPC 30l 20l 17
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
PCR autoclaving tape 200 hr 200ml OCT compound -80 30m 1 1 RNase-Free MQ wash5 0.03% 5 DEPC-MQ wash 2 70% 1 OK DEPC-MQ Cool 5-80 19
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 20 80 over night 10.8l RNase-Free Water 20
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
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. 2002 Jan;131(1 Suppl):S39-47. 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. 2003 Feb;442(2):159-62. 22
40%70% 70%,40% 23
40% 40% 24
25
SP 10,000/SEC n- -79 n- n- n- n- n- -95 26
SP bp. Boiling point mp. Melting point bp. mp. () Water 100 0 100 (Helium) -268.9-272.2 3.3 (Nitrogen) -195-210.01 15.01 (Oxygen) -182.96-218.4 35.44 (Ethane) -88-172 84 (Propane) -42-187.7 145 (Isopentane) 27.85-159.9 187.75 12(Freon 12)* -29.79-158 128.21 (Acetone) 56.5-94 150.5 n- (n-hexane) 69-95 -100 164 * 12 2000 10-6K/SEC 10-4K/SEC (1,000,000/SEC) (10,000/SEC) CE* ETHANE -172 1.3 LN2 at RT*(BILING) -195 0.1 LN2 COOLING to FP*(SLUSH) -210 0.2 ISOPENTAN -160 0.5 FREON 12-158 0.5 * CE (LN2) (Ethan)1.3 0.5 * RT Room Temperature * FP Freezing Point 27
SP dyne/cm 72.7 48.4 35.2 30.2 (o,m,p) 30.0, 28.6, 28.3 n- 25.2 (MKE) 24.6 n- 24.6 24.0 24.0 (MIBK) 23.6 22.6 18.6 28