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RT-GPCR TechNote -RT-qPCR DAA|B"Y BAlS 2012.09.20 sts Sat 1 Life science Workflow 2. qPCR (Real-Time PCR) 9| glajgt S44 3. PCR 2b qPCR 9} fo} 1. gee w Bel 1. RT-gPCR Workflow 2.RNA #2] : a 2 @ RNA prep Al FolAte @ Prep @ RNA qual 3. cDNA oh © RT primer. @ATkit 4 4. qPCR © aPCR as 2 Baas ey @ Primer design @ Reaction ©174 Oe4 svn sons sone . HOB 712] Alor RT-yPCR I. Introduction LLlife science WorkFlow = Genomics s = Sample preparation RNA purification s s Characterization T-PCREGPCR, 2.qPCR (Real-Time PCR) | gizl9} $2 ual a2] »> PCR SS S}01.A4BOl starting DNA or RNA concentration 440] Saf “PCR2| Zale log phase BIZ SOME vel sta] SHEL. POR @ St SY SHS SHE YA St B Sdo2 wo! 3. PCR} qPCRO| Ato] - REPCR TgPcR A ___| nd Point Plateau Phase Logarithmic Phase BAZ anda! intensity VOR we Anpcation to} | SEAS a 4 SUE BUI Se Pegs = wee dEC Boe as Fe 8 : zea a See Bea | aeea wuss 9319 Be apes za | gerecr ge, ack os e | SLB DYX|AY BAS 2012.09.20 Frmwsaswee 1 RT-gpcr 1. RT-qPCR WorkFlow gta: RNA Se] |{_ 2eH7]: cDNA BA 3Eb aI: PCR BBE el mRNA Se seat RNA HBR Ei Primer B AB || cONA OL space primer 1 ti ao] uo 8 foal RNA] | of CONA Bae we so ree Bee Be BS, oe ue janes © Specific Primer Cx}! 1] Standard curve test QPCR YS © hn ee Fe |e RT esol ey | RNA ## Reagent AHS || @ Primer “79 | it ANB | Leute pees | | 2. RNA wal 0 Heese RNA prepA| FOIA RNA EDNAN O88 HINZE OF! single sande) OPE! 2eH0|7] cHBO|Degradationo| @ AA ojetch. O18 waisi2] sHe! Fests] sc -prep 2 RNA prepol A-BSI& 2E BZ, 7171, pipette SS 70% ONE O1BM St Of RNase SCHR! HA, “tubes and pipette tins & RNA prep O| A'BSIe ZE MBS Nuclease-free ab ware 248 ANALE 28 AISI BSR BOL HEAL latex glove HE j-RNasetree reagents AS (0f, Water, nuclease-free, #ROSBL). ~ Apechyly yncor tw te -RNase inhibitor A} (0, FermentasRiboLock™ RNase Inhibitor, KO731 Kit Oh) column based DNAVRNA extraction kit APB @Prep (Kit Qoj(TrizolS 018! manual w= BB) RNA purificatiStrKit (Fermentas cat#KO731) Iysate eee Ethanol 271 ° bSolutiong column. Bich ~_ shiver. th . Centrifuge Imimch#e! Boh alah He 184 centrifuge (min) MBE tube colume 8: ‘Add wash butfer Centrifuge Imimc}@e! Sot wHepelgl cdAddwash buffer Cenviuge 2mimcreel Boh chet Oar at be. eMBE tube@ columng $2}. ‘Add 50-100 ul Centrifuge trim Fhe lease free water DNase I #42] (Fermentas cat#EN0525) Total RNA {Bo genomic DNA} ZEISIO} gl Rol RAC}. Genomic ONAE PCR] FeO} SF 71 BOL O}e! ARE OBA os BAPE BAEC gl > aICt Total RNAB 8 , DNase I (RNase-free) AHBSIO} genome DNAB ¥aHs!ch. DNase 1 2] F, EDTA 2] & 651C Smin heating B10} enzymes inactivation AIZICt. @ RNA quality check “A260/280 ratio: 18-20 Mole L}2/0} ANA gelOl loading $82 ti, 285 9 1857 smear 3X) 22, J=2es BoP 2 band 7} UEILION ses (loading | trayS} butters Fresho}7t A+B) ‘integrity 285 +188 = 15-20 Of) 22% AH{I9} TELS ANA ladder. 241 lane degradation S! AfeH. 2 IML 2 band} S215IO}0} Bh SUB BARE BAS 2012.09.20 3. DNA ta +B2]S) RNAEME handlingd| 01217] CHRO Reverse transcriptaseZ O|B3} 2S ONAE HHFE We MONG Kite] AP, UY FASS mix slo] US + US) Template RNA (total RNA SE mRNA) god, Random, SS Primer) ‘AT UB BA (EE 2S, Modified MMLY) ante RNase Inhibitor “RT Buter Primer DRT primer -Random pe ‘Qult21 Az} wal B|ALOLS random primer AHSLE Hol Sct, Random primer Abe MRNA Ba] WOOL AM SSHSS AWA VBS + Slo} PCR SF esI04 or) a WH AB eFeobT LAB + RIC. andom hexamer NLEe FOI7} w 284CH) 500 bp O|a42] FYOINE 77] WHO] SURE BPS Vb -PCRO| 4 CIM: Real Time PCR target SH size’ VE 300 bp olsjolae sz ySe ae ac Annealing G1 (3 step PCR) : BESO WE APE SSS OAL AIS BA AS OC. SERS] UE ASE SEE UAL AE BA Sect “MB WS EA G step PCR): AB AIS ZA SBOE MIRO SHBO] 97171 1F2 2 FOV BazCh, -ycle# = AZOLE 40 cleo AID. Bool chet qlee Seech Oe cia ear Ctat HE Yeole VSS ore ALo] O47} (Threshold)s} SH SMo| DAS CULE se Bel (Crossing Point) lol, SS AAS 2 SerES Fo} 2210] a C47t SLE BS CewOS SHE YO} ic} (2nd Derivative Maximumy) +Standard Standard sampleo| 14% S|4o] Olof BS + ot ARS o|x| Bs] Bol > S 7S SF fle}, PCR SH BOWL} As eet wa S Se WE We + Ch UMZ ABMS WS oy, GA 5-6 eta} SEO} standard samples Zulge Ch. O| sampleS BLS Real Time PCRE BAlSH 212 SSO} CHS AMBEICH. creat 2} £7] FB SES] FALE Bell Bol Wook ARIS Ase + ach SlB RT-qPCR BAYAAY BAS 2012.09.20 Haeenswmee we * BO BB RE MES 18 AIMS Yael ON BES BawE Susie wy “Reference gene : GAPDH BURcopy4)0] 71F2e On| BBM VO MAS 7H! RE Yeo] Beajcy “Target gene : p53 “Standard curve 414 57H O[8, SompleO| standard curve LEO} 20/0} etCh uplcations (#3, #2) a [Sample cess (CE GAPDH ApS3-GATAACt [2A-AACt [Rel to p53.SIRNA non-target siRNA wes] 2aa3|_—_8a2|__o00)2*-00 3.00 53 siRNA aise] 2915] i2asl_30a[2--361 ‘osi852 2 sample [ee ps3 [ck GAPDH Apsa-GAtaact [2-aACt [Rel. to p53 siRNA non-target RNA sim asarl__825| 000 5-000 1.00 [p53 siRNA —_ #223] 3042] i180 355 fn-ass 085522 Initial Quantity’ Ct value +/ $0. 108 24.85 +/.0.06 fo 28.44 4/-0.01 * Copies unknown 26.76 = 3408 copies HBS: Al BIS KUT We MSS Woe ogsin VE SETS] Wao] SIAL ABH chet control sampleOl THOM WOH SSD EAS ASHE eeYoict. eH BB MWIME BAIS UD Ye Sy RAT Sol WEA reference SUN AVS AAI BIC. Reference RUTH: MBS FHF REIS oe Aol, Sezt wel HHA VEILS AUWASR housekesping FUR 1B EICt ‘Reference gene B®. (GAPDH oF Bractin) abel Using absolute quant Healy ERBB Care ERB? ——————— Healthy GAPOH Lobel Normalization Featty 2479527000 Carcinoma _16363/40276 met ela gay ERBEE | ERB2 _GAroH EMBED: (eathy 25 se OT Coe ENS 5417 Rel. to p53 siRNA Rel. to p53 siRNA [Wa [BR 1.00) 100] 100 (0.081952 0085522| 008 120 Relative mRNA transerits BB g on-target sina 53 saNAa RT-qPcR Thermo Scientific PikoReal Real-Time PCR System [ Phchen 9 Rete BER Sytem eserpton PhoReal24 Rea Tine FR Sytem i Piko® Thermal Cycler Specifications Size [16 cm wide, 17 cm deep, 23 cm high lWeight [4 kg (with extemal power supply) Power supply Universal put with maximum draw of 180 W_ Block Configurations [24-well and 96-well Max instantaneous ramp rate ln excess of 8°C per second Max average ramp rate 5*C heating and 4.5°C cooling [Thermal uniformity +/- 03°C [Thermal accuracy /- 0.2°C [Thermal range lorc-99.9°¢ [User interface [Semi-graphical programming Number of programs lover 1 000) Heated lid Fully automated and motorized function Warranty 2 years parts and labor m. Boe 7171 a alg “Thermo HE RT ‘Thermo Scientiic Maxima RT ‘Thermo Scientific RevertAidPremium(not Thermo Scientific Phusion ins) RevertAidand RevertAid H Minus (H-notin US) Thermo Scientific Verso SlB DAE BAS 2012.09.20 Frmessose PCR Standard: ‘Scientific DreamiTag Thermo ScientficTaq Z Hotstart: ‘Thermo ScientiicPhire HS I ‘Thermo Scientiic Maxima HS High fidelity: Thermo Scientific Phusion & Phusion HS Direct PCR: Kits trom various sample materials SLB RT-qgPCR DAA AY BAS 2012.09.20 Samencwees -QPCR 712/% reagent Sets Pere Pr eae . . . . & . . . . . * . . . . H Ff ° € . ° . . ° . . ° . . . . ° ° . . . . . 3 . . . . . ° . . . . FH 2 . ° . ° . ° . . ° . . . . . ° . . . . . . . . . . . . . . . . . . a ee a a Pk Therma —_ sl os 7 aes Finnzyme 7eP0036, ‘Piko® Thermal Cycler S6-well systern uss 7a Pre NA Puro rae0t Gee at A Pieton WR NA Patton oT Gene ANA uicton Fementas [“xo7a1 | GeneeT= Whe od ANA Preton RE Srna Viel RNA Min e525 Rae Rise fre sopping wit Wa Twa teew | Reman | —swn651 Tr NA Leos iAtoadig Oy | Femenas | — mse A ig use| 7500 Tatiana syne Fenayne [Fare Oy DNAS sere isene | A055 cDNA Sti vast Fe Sd Sy asst fest re fermenas | Tee Ta et Sand ONA Sy Sosa waa Fee cHo™ Fe Sand DNA Steve Dresct erara ann Reese Tnserpase 20 ni sone OigesT8 ier Tul sour Fandom Hear Pr Ho fermenter [nn GNF 10 ech Ta AT supplement -ENO201 RNase H 100 units EL Tk Rn a Riese fxorsnend 5-559 | Sant PR ve Gren Non x pee Va | NTA re-deignes sagene | A8:951 | Sos PCR Gre presi ae ixrcicirg ROH | NTE AS52 | Sas gC eve on ar nung on ROX | NTA omar LESH ara Fob PCR ae oe stundwa OIST | Wana rab PR aia 9, Oxon pod | ROR QUST GPA ASTER aE 75860 | Vague Fab PX ir No ters 585 Dye Colao rake pc Sire ae Fass _DyNamo Flash Probe gPCR Kit SAUL Probe fest use 75680 “WeriQuest Fast Probe QPCR Master Mix (2X) 75685 | "Vig fo rb PR nn No AterceR 75-300 eno Step FR 7 regen [Taser Yao Se GRACE ROL Ne ate Ree A907 Ya Sep PCR th ow HE 7570 VERQUEST PROBE ONESTEP QUTPCR oe 75710, VERIQUEST PROBE ONE-STEP QRTPCR, NO DYE aaa Tins SYR GeV AOX CRN i aaa Famers [Atos — | Wana 58 Gren PER en HD [ wT staaars 0251 | Waine® SER Geen PER Men, ROX hon prove] — ROU oe seo ‘ERGUEST STOR GREEN GPCR 7565 | _WenGoeSYR en ER ses wih Fron Fas Dye Cole SER Gen PCR sie rest ee FAls _DyNAmo Flash SYBR Green GPCR Kit SAIKL ‘svBR " uss. 75690 VeriQuest Fast SYBR Green qPCR Master Mix (2X), a AB-4104 ‘Verso SYBR Green 1-Step QAT-PCR kit plus ROX vial A os rans en Sp TPR SYR er AOE NE tepargeen | [Tassie eso 1 Step QRF-PCR SYBR Geen Low 8OK Mi Ae fas) eo Sp FR SYR Grr Fen Mn a re eG SBR Geen Onesie 75715 | WRIT TR GEN ONESTPGRTOCR wT HOUR tly TBlcle- web kk BReker oz'6ozt0z As

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