Real-Time PCR
DNA Real-time PCR Exicycler 96 RotorGene Q/6000 PCR HPV16 HPV18
Mix1 Mix2 /μl /μl /μl /μl /μl C 1 : International Unit 2 Lot# 3 Freeze & thawing
1 MSDS: Material Safety Data Sheets
(TaqMan Real-time PCR HPV16 ' FAM HPV16 BHQ1 TexasRed PCR ' ' ' JOE HPV18 BHQ2 BHQ1 HPV18 ' ' in vitro in vitro PCR 1 Validation 2 Conserved 3 Beta2 Microglobulin
PCR Real-Time PCR Real-time PCR PCR Real-time PCR PCR PCR PCR Real-time PCR SYBR Green DNA DNA DNA 1 Post PCR 2 UV Transilluminator 3 Non Specific Methods 4 Signal
PCR TaqMan Real-time PCR MRD 1 Specific Methods 2 Minimal Residual Disease
HPV18 HPV16 HPV18 HPV16 HPV DNA HPV16 HPV18 1 High-risk HPVs 2 Precancerous 3 Screening 4 Cytology 5 High-grade cervical disease 6 Grade 7 Squamous intraepithelial lesions
/l HPV18 HPV16 HPV18 pvalue HPV16 /l HPV18 /l HPV16 1 Analytical Sensitivity
Varicella Zoster Virus Human Cytomegalovirus 2nd WHO International Standard for Hepatitis B DNA for Nucleic Acid Amplification (NAT) Assay Epstein-Barr Virus 2nd WHO International Standard for Parvovirus B19 DNA for Nucleic Acid Amplification (NAT) Assay WHO 2nd International Standard For Hepatitis C Virus RNA for Genomic Amplification Technology Assays Herpes Simplex Virus Type1 Herpes Simplex Virus Type2 (DNA) 1 Analytical Specificity 2 Cross Reactivity
HPV16 /µl HPV18 HPV16. HPV18 /µl /µl 1 Linear range 2 Precision 3 Variance 4 Intra assay variation 5 Inter assay variation
/µl /µl PCR. C t /µl Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance Exicycler96 HPV16 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance Exicycler96 HPV18 1 SD: Standard Deviation 2 CV: Coefficient of Variation 3 C t : Cycle of threshold
Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance Exicycler96 HPV16 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance Exicycler96 HPV18 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance RotorGeneQ HPV16
Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance RotorGeneQ HPV18 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance RotorGeneQ HPV16 Ct Ct CV Ct CV Intra Assay variation Inter Assay variation Total variance RotorGeneQ HPV18
Real-time PCR (Heating-Block) DNA rpm Real-time PCR PCR PCR 1 DNase RNase Free 2 Vortex 3 Spin 4 Strip 5 Polymerase Chain Reaction (PCR)
C C C C 1 CytoBrush 2 Mucolytic 3 Urethral epithelial swab
dntp PCR Mix1 Mix2 HPV18 HPV16 PCR PCR R 2 R^2 e PCR 1 Efficiency (e PCR )
DNA (Cat#) Qiagen 51104 DynaBio Blood/ Tissue Genomic DNA Extraction Mini Kit QIAmp DNA Blood Mini Kit 1 Cross Contamination
1 Sampling 2 Under or Over estimation 3 Duplicate Real-time PCR
PCR x x Real-time PCR 1 Run
PCR Exicycler96 (Scan) HPV16 FAM HPV18 JOE TexasRed RotorGene 6000 RotorGene Q Orange Green Yellow (Scan) HPV16 FAM HPV18 JOE TexasRed
ExiCycler96 Design Experiment Exicycler Run File a. Master HPV16&18 b. Protocol Takapouzist c. Plate 920101 /.,
Append Incubate Temperature: 95 Temperature: 95 Temperature: 56 Time: 5 min Append Time: 15 sec Append Time: 50 Sec Append Temperature: 72 a. Line=2 b. Cycle=50 Append Scan Incubate Time: 20 Sec Append Go to
Append Plate Add Probe.1 JOE BHQ FAM BHQ.2 OK TexasRed BHQ
E5 B5 B5 E5 Probe Name Assign STD FAM JOE Type Sample TexasRed Sample Name Assign Sample Name
OK Plate Protocol Save Exicycler3 Run Master save as
.8 Run Exicycler Master Design Experiment (open) Door Run.9.11.11 OK
RotorGene Q RotorGene 6000 New Locking Ring Attached New Quick Start 36-Well Rotor Next Next PCR
Gain Optimisation Auto-Gain Optimisation Set Up OK Green Auto-Gain Optimisation Set Up Add
Add Target Sample Range Ok Yellow Auto-Gain Optimisation Set Up Add
Target Sample Range Ok Green Add Orange Target Sample Ok Yellow Green Range
Perform Optimisation Before 1 st Acquisition Close
Next Start Run
OK RotorGene 6000 /µl RotorGene Q
File Exicyler96 Exicycler Run Exicycler Analysis Open Open Absolute Quantification Ok
Trend Average Sub. Method Threshold Level Flu. Graph TexasRed FAM JOE STD Curve R^2 Efficiency Ct Report Form
RotorGeneQ RotorGene6000 Analysis Analysis Show
Dynamic Tube Slope Correct Auto Scale Linear Scale Threshold Maximize
Quantitation Analysis Ct Standard Curve R^2 Efficiency Quant. Results HPV18 JOE Orange
HPV16 TexasRed FAM HPV16 HPV18 TexasRed JOE HPV18 JOE FAM HPV18 HPV16 TexasRed HPV18 HPV16 JOE FAM 1 Flowchart 2 Trouble Shooting
TexasRed C t > JOE FAM PCR HPV18 HPV16 TexasRed JOE FAM
HPV16 Texas R^2> Ct FAM JOE FAM JOE FAM PCR HPV16 Texas
HPV18 Texas R^2> Ct JOE JOE FAM JOE FAM PCR HPV18 Texas
/l Result (/ml) = Result (/µl) Elution Volume (µl) Sample Volume (ml) /µl (/ml) = /ml R^2 e PCR 1 PBS
() /ml /l HPV16 WHO WHO HPV18 1 WHO: World Health Organization 2 Viral Loads 3 1 st WHO international Standard for Human Papillomavirus (HPV) Type16 DNA, code: 06/202 & 1 st WHO international Standard for Human Papillomavirus (HPV) Type18 DNA, code: 06/206 4 Genome equivalent
Symptom JOE FAM JOE HPV18 FAM HPV16 PCR TexasRed PCR PCR PCR JOE FAM PCR PCR TexasRed JOE FAM DNA
Symptom PCR PCR FAM JOE FAM TexasRed JOE PCR PCR Real-time PCR
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