鶏骨格筋から分離した単一筋線維の筋線維型判別方法の開発
Contents
1. Fig 2B 4 PFA 4 C 24 Fig 2 A si2. cDNA 1 sTo mRNA 1I fT mRNA 20 6 sT 20 6 fTnI RNA Fig 3 sTnl fTnl GAPDH PCR cDNA PCR 4 PFA 4 C 24 0 2 37C 6
3. 4 PFA 40 NaOH RNA RNA RNA RNA Fig 1 A single fiber isolated by using 40 sodium hydroxiside solotion PBS Fig 2A 24
4. E mRNA X mRNA 1 1 Zev UAB Po bet As RE
5. SZ4045 Dumont Montignez AX80 Switserland H RNA 2 5 0 2 0 2 0 2 0 2 1mL 2 0mL 37 C 6 MAE LIX RNA E
6. 24 PBS pH 7 2 4 PBS 4 PRU OE Lh Cbs 4 C 24 PBS 4 2 4 40 NaOH Wada 2002 40 NaOH ImL 2 0mL 3 Bil BRE
7. RNA PCR E Glyceraldehyde 3 phosphate GAPDH 1I E 4 2 D E 2
8. RNA C D Data Rn vs One Fig 3 Results of real time PCR Slow type troponin I A B Fast type troponin I C D One cDNA was used for A and C and another cDNA was used for B and C 4 5 1 2 3 4 5 6 Barnard RJ Edgerton VR Peter JB Effect of exercise on skeletal muscle I Biochemical and histochemical properties Journal of Applied Physiology 1970a 28 762 766 Baldwin KM Klinkerfuss GH Terjung RL Mol PA Holloszy JO Respiratory capacity of white red and intermediate muscle adaptative response to exercise American Journal of Physiology 1972 222 373 378 Hirabayashi M jiri D Kamei Y Tajima A Kanai Y Transformation of skeletal muscle from fast to slow twitch during acquisition of cold tolerance in the chick Endocrinology 2005 146 399 405 Nishida J Machida NW Tagome M Kasugai Y Existence of parvalbumin and biochemical characterization in quail and pigeon skeletal muscles with different fiber type compositions Journal of Experimental Zoology 1997 277 2
9. 2 2 1 2Hz0 12HzO Germany Darmstadt Gibco grand island NY USA NucleoSpin RNA XS kit Prime Script RT reagent Kit SYBR Primix EX TaqTM E ReliaPreTM RNA Tissue Miniprep System Promega Madison WI USA greiner bio one Frickenhausen Germany Ross308 3 p E 1 24 22 3 1Mcalkg ees BHR E Ls 25 1 C amp UTE FIC 1 14
10. 2 6 RNA RNA NucleoSpin RNA XS kit ReliaPreTM RNA Tissue Miniprep System 100uL Buffer RAI 2uL TCEP NucleoSpin RNA XS kit 250uL LBG TG Buffer ReliaPreTM RNA Tissue Miniprep System 1 5 mL RNA 2 7 cDNA NanoDrop Lite Waltham MA USA Prime Script RT reagent Kit cDNA 2 8 Real Time PCR PCR 1 mRNA 7300 Real Time PCR system Applied Biosystems Foster City CA PCR 50 C 2 95 C 2 95 C 1 55 C C15 72 C 1 70 Table 1 List of primers sequence used for quantitative real time polymerase chain reaction Gene sTnl Forward Reverse fTnI Forward Reverse GAPDH Forward R
11. 83 292 Wada KI Takahashi H Katsuta S Soya H No decrease in myonuclear number after long term denervation in mature mice American Journal of Physiology 2002 283 C484 488 Jemiolo B Trappe S Single muscle fiber gene expression in human skeletal muscle validation of internal control with exercise Biochemical and Biophysical Research Communications 2004 320 1043 1050 7 Daichi Ijiri Moe Miura Yukio Kanai Miho Hirabayashi Increased mass of slow type skeletal muscles and physiologically depressed myostatin gene expression in cold tolerant chicks Zoological Science 2009 26 277 288 Identification of muscle fiber type from single muscle fiber in chicken skeletal muscle Daichi Ijiri Department of Biochemical Science and Technology Kagoshima University 1 21 24 Korimoto Kagoshima 890 0065 Japan TEL 099 285 8652 The aims of this study was to isolate single muscle fiber from chicken skeletal muscle and to distinguish muscle fiber type The sartrius muscle collected from chicken leg was incubated in 4 Paraformaldehyde solution in PBS at 4 C for 24 hours Then it was digested in 0 2 collagenase solution at 37 C for 6 hours and single muscle fibers were isolated by using tweezers under stereoscopic microscope Total RNA was purified from each muscle fibers and real time PCR was performed Glyceraldehyde 3 phosphate dehydrogenase mRNA was amplified in all samples And muscle fiber type of single fiber was indentified by usi
12. Ze 1 J H 21 24 T 890 0065 TEL 099 285 8652 RNA HEL SNR Re BCH iit ERICE cDNA dehydrogenase 1 ROA AHA 4 4 C 24 0 2 37 C 6 E
13. everse Sequence 5 3 Size BP Accession No GAC CTG AGA GCC AAC CTG AA 175 XM 419242 3 TTC TGT CCT GGT GCT CCT CT CGG ACT CGG GTT CAC AAC CA 108 NM 205417 1 GCA AGC TGG AGC ATA GCA CT CCT CTC TGG CAA AGT CCA AG 200 NM 204305 1 CAT CTG CCC ATT TGA TGT TG sTnl slow type troponin I fTnl fast type troponin I GAPDH glyceraldehyde 3 phosphate dehydrogenase 3 PBS 40 NaOH 4 PFA 4 C 24 40 NaOH Fig 1 4 PFA 40 NaOH 4 PFA 40 NaOH
14. ng real time PCR
15. ngle fiber isolated by using enzymes A 0 2 collagenase solution B 0 2 collagenase with 0 2 dispase solution 4 PFA 4 C 24 RNA NucleoSpin RNA XS kit ReliaPreTM RNA Tissue Miniprep System 10 RNA RNA NucleoSpin RNA XS kit 112 59 46 83 ng uL n 10 ReliaPreTM RNA Tissue Miniprep System 12 38 6 27 ug uL n 10 RNA By OO SI glyceraldehyde 3 phosphate dehydrogenase GAPDH RNA cDNA GAPDH PCR 20 20 JV RNA IX PCR
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