PrimeScript™ RT reagent Kit (Perfect Real Time)

1 RR037A primescript RT reagent Kit (Perfect real time ) v201903Da RT-PCR primescript RTase PCR cDNA 2 RT-PCR PCR TB Green Premix Ex Taq II (Tli RNaseH Plus) RR820S/A/B 1 TB Green Fast qPCR Mix RR430S/A/B 1 TB Green Premix Ex Taq (Tli RNaseH Plus) RR420S/A/B 1 TB Green Premix DimerEraser ( perfect real time ) RR091A/B 1 PCR Probe qPCR Mix RR391A/B PCR TB Green 1 PCR qPCR 2017 10 TB Green I 200 10 l 1 5 primescript Buffer (for real time ) 2 400 l2 primescript RT Enzyme Mix I 3 100 l3 Oligo dT Primer 50 M 100 l4 Random 6 mers 100 M 400 l5 RNase Free dH2O 1 ml6 EASY Dilution (for real time PCR)

2 4 1 ml 2 dNTP Mixture Mg2 3 RNase Inhibitor 4 total RNA cDNA TE EASY Dilution (for real time PCR) PCR PCR EASY Dilution (for real time PCR) 9160 EASY Dilution (for real time PCR) PCR 37 C 42 C 85 C ml ml II 20 C2 RR037 AIII 1 15 PCR cDNA 2 RT-PCR 2 Random 6 mers Oligo dT Primer Gene Specific Primer 3 TB Green PCR TB Green Random 6 mers total RNA 4 RT-PCR total RNA cDNA

3 TE EASY Dilution (for real time PCR) IV 1 10 Master Mix RNase Free dH2O Master Mix 2 primescript RT Enzyme Mix I 50 3 3 RR037AV RNA VII-B. RNA 10 TB Green Assay 1 RNA RNA 1 5 primescript Buffer (for real time )

4 primescript RT Enzyme Mix IOligo dT Primer 50 M 1 Random 6 mers 100 M 1total RNARNase Free dH2O2 l1 25 pmol50 pmolTotal10 l 2 1 Oligo dT Primer Random 6 mers mRNA cDNA Gene Specific Primer Oligo dT Primer 50 M Random 6 mers 100 M Gene Specific Primer 2 M l25 pmol50 pmol1 pmol 2 10 l 500 ng total RNA 2 37 C15 3 85 C5 4 C 3 Gene Specific Primer 42 C 15 PCR 50 C 2. PCR PCR 10 Probe Assay PCR TB Green 4 RR037A Probe Assay 1 RNA RNA 1 5 primescript Buffer (for real time )

5 primescript RT Enzyme Mix IOligo dT Primer 50 M 1 Random 6 mers 100 M 1total RNARNase Free dH2O2 l2 l1 25 pmol200 pmolTotal10 l 2 1 Oligo dT Primer Random 6 mers mRNA cDNA Gene Specific Primer Oligo dT Primer 50 M Random 6 mers 100 M Gene Specific Primer 2 M l2 l25 pmol200 pmol1 pmol 2 10 l 1 g total RNA 2 37 C15 3 85 C5 4 C 3 Gene Specific Primer 42 C 15 PCR 50 C 2. PCR PCR 10 TB Green Assay 10 l 500 ng total RNA 5 RR037 AVI PCR TB Green Premix Ex Taq II (Tli RNaseH Plus)

6 RR820A PCR Thermal Cycler Dice real time System II 1 PCR 1 TB Green Premix Ex Taq II 2 l1 PCR Forward Primer 10 M 1 M 1 PCR Reverse Primer 10 M 1 M 1RT cDNA 22 l lTotal25 l 3 1 M M 2 total RNA 10 pg 100 ng cDNA template PCR 10 3 : 25 l 2 PCR PCR PCR Tm PCR 3 PCR TaKaRa Ex Taq HS Taq PCR PCR PCR 95 C 5 15 PCR 95 C 30 3 Thermal Cycler Dice real time System PCR Hold Cycle 195 C 30 2 Step PCRC ycle 4095 C 5 60 C 30 Dissociation6 RR037A Applied Biosystems 7300/7500/7500 Fast real - time PCR System StepOnePlus real - time PCR System

7 1 PCR 1 TB Green Premix Ex Taq II 2 10 l25 l1 PCR Forward Primer 10 M l2 M 1 PCR Reverse Primer 10 M l2 M 1 ROX Reference Dye 50 or Dye II 50 2 l1 l1 RT cDNA 32 l4 l 6 l16 lTotal20 l 450 l 4 1 M M 2 ROX Reference Dye II 50 ROX Reference Dye 50 Applied Biosystems 7500/7500 Fast real - time PCR System ROX Reference Dye II 50 StepOnePlus 7300 real - time PCR System ROX Reference Dye 50 3 20 l total RNA 10 pg 100 ng cDNA template PCR 10 4 2 PCR PCR PCR Tm PCR 3 PCR Applied Biosystems 7300/7500 real - time PCR System StepOnePlus PCR Stage 1 Reps 195 C 30 Stage 2 PCR Reps 4095 C 5 60 C 30 34 Dissociation Stage StepOnePlus 30 7300 31 7500 34 7 RR037A Applied Biosystems 7500 Fast real - time PCR System PCR Holding StageReps 195 C 30 Cycling StageNumber of Cycles 4095 C 3 60 C 30 Melt Curve Stage TaKaRa Ex Taq HS Taq PCR PCR PCR 95 C 5 15

8 PCR 95 C 30 3 PCR 8 RR037 AVII AppendixA cDNA primescript RT reagent Kit ( perfect real time ) total RNA 2 pg 2 g 20 l Random 6 mers 37 C 15 30 60 85 C 5 4 C PCR TB Green Premix Ex Taq ( perfect real time ) 2 l 25 l Actb perfect real time Thermal Cycler Dice real time System LegendTimeRSqEff Standard Curve 15 LOG X 30 LOG X 60 LOG X 15 30 60 15 30 60 9 RR037AB RNA RNA RNase RNase RNA RNase RNA 1 2 1 180 C 60 2

9 DEPC 37 C 12 DEPC 120 C 30 RNA RNA 180 C 60 DEPC DEPC RNA RNA RT-PCR RNA RNA GTC RNA total RNA NucleoSpin RNA AGPC RNAiso Plus 9108/9109 DNA total RNA DNA DNA PCR DNA total RNA 1 DNA 2 DNase I DNA 1 DNA DNA DNA 2 DNA PCR mRNA 2 DNase I 10 RR037A 2 DNase I DNA total RNA Recombinant DNase I RNase-free 2270A DNA DNase I [ ]

10 1 total RNA20 50 g10 DNase I Buffer5 lRNase Inhibitor20 U DNase I RNase-free 2 l 10 U DEPC 50 l fill up2 37 C 20 3 DNase I A 1 l M EDTA 80 C 2 2 DEPC 100 l fill up B 1 50 l DEPC 100 l 25 24 1 2 15,000 rpm 5 3 24 1 4 15,000 rpm 5 4 10 l 3 M 250 l 10 5 4 C 15,000 rpm 15 6 70 4 C 15,000 rpm 5 7 8 DEPC DNA PCR