RT 2 Profiler PCR Array: Web-Based Data Analysis Tutorial
|
|
|
- Horatio Nelson
- 8 years ago
- Views:
Transcription
1 RT 2 Profiler PCR Array: Web-Based Data Analysis Tutorial Samuel J. Rulli, Jr., Ph.D. qpcr-applications Scientist Samuel.Rulli@QIAGEN.com
2 Pathway Focused Research from Sample Prep to Data Analysis! -2-
3 Topics to be Covered. Topic I: Brief Technology and Protocol Overview -Offered every Month! (Next RT 2 PCR Array Webinar) Topic II (Today): PCR Array Data Analysis Defining Baseline and Threshold Web Portal Location / Address Uploading Raw C t Data Analyzing Data & Controls Exporting Data -3-
4 Anatomy of a PCR Array. 84 Pathway-Specific Genes of Interest. 5 Housekeeping Genes. Genomic DNA Contamination Control. Reverse Transcription Controls (RTC) n=3. Positive PCR Controls (PPC) n=3-4-
5 Anatomy of a PCR Array. 84 Pathway-Specific Genes of Interest. 5 Housekeeping Genes. Genomic DNA Contamination Control. Reverse Transcription Controls (RTC) n=3. Positive PCR Controls (PPC) n=3-5-
6 How RT 2 Profiler PCR Arrays Work. cdna Synthesis (C-03 kit) Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.). Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet. Run 40 cycle qpcr Program Standard cycling conditions for all Real Time PCR Instruments 2 hours. Upload and Analyze Data (FREE) 15 minutes from Raw Ct to Fold Change Data -6-
7 How RT 2 Profiler PCR Arrays Work control Hot Sauce. cdna Synthesis (C-03 kit) Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.). Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet. Run 40 cycle qpcr Program Standard cycling conditions for all Real Time PCR Instruments 2 hours. Upload and Analyze Data (FREE) 15 minutes from Raw Ct to Fold Change Data -7-
8 How RT 2 Profiler PCR Arrays Work control Hot Sauce. cdna Synthesis (C-03 kit) Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.). Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet. Run 40 cycle qpcr Program Standard cycling conditions for all Real Time PCR Instruments 2 hours. Upload and Analyze Data (FREE) 15 minutes from Raw Ct to Fold Change Data -8-
9 How RT 2 Profiler PCR Arrays Work control Hot Sauce. cdna Synthesis (C-03 kit) Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.). Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet. Run 40 cycle qpcr Program Standard cycling conditions for all Real Time PCR Instruments 2 hours. Upload and Analyze Data (FREE) 15 minutes from Raw Ct to Fold Change Data -9-
10 How RT 2 Profiler PCR Arrays Work control Hot Sauce. cdna Synthesis (C-03 kit) Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.). Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet. Run 40 cycle qpcr Program Standard cycling conditions for all Real Time PCR Instruments 2 hours. Upload and Analyze Data (FREE) 15 minutes from Raw Ct to Fold Change Data -10-
11 How RT 2 Profiler PCR Arrays Work control Hot Sauce. cdna Synthesis (C-03 kit) Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.). Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet. Run 40 cycle qpcr Program Standard cycling conditions for all Real Time PCR Instruments 2 hours. Upload and Analyze Data (FREE) 15 minutes from Raw Ct to Fold Change Data -11-
12 How RT 2 Profiler PCR Arrays Work control Hot Sauce. cdna Synthesis (C-03 kit) Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.). Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet. Raw C(t) Values. Fold Change Results Using C(t) calculations ABL1 is up/down regulated by x fold -12-
13 How RT 2 Profiler PCR Arrays Work control Hot Sauce. cdna Synthesis (C-03 kit) Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.). Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet. Raw C(t) Values. Fold Change Results Using C(t) calculations ABL1 is up/down regulated by x fold -13-
14 Topics to be Covered. Topic I: Brief Technology and Protocol Overview Thursday January 6 1pm EST (Next RT 2 PCR Array Webinar) Topic II (Today): PCR Array Data Analysis Defining Baseline and Threshold Web Portal Location / Address Uploading Raw C t Data Analyzing Data & Controls Exporting Data -14-
15 Topics to be Covered. Topic I: Brief Technology and Protocol Overview Thursday January 6 1pm EST (Next RT 2 PCR Array Webinar) Topic II (Today): PCR Array Data Analysis Defining Baseline and Threshold Web Portal Location / Address Uploading Raw C t Data Analyzing Data & Controls Exporting Data -15-
16 Defining Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:. Baseline Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15). Threshold Value Use Log View Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve. Export C t values to blank spread sheet (Excel).. Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) ) *For Roche LC480: Use Second Derivative Maximum -16-
17 Defining Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:. Baseline Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15). Threshold Value Use Log View Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve. Export C t values to blank spread sheet (Excel).. Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) ) *For Roche LC480: Use Second Derivative Maximum -17-
18 Defining Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:. Baseline Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15). Threshold Value Use Log View Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve. Export C t values to blank spread sheet (Excel).. Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) ) *For Roche LC480: Use Second Derivative Maximum -18-
19 Defining Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:. Baseline Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15). Threshold Value Use Log View Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve. Export C t values to blank spread sheet (Excel). Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) *For Roche LC480: Use Second Derivative Maximum -19-
20 Defining Baseline and Threshold *For Roche LC480: Use Second Derivative Maximum. Baseline Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15). Threshold Value Use Log View Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve. Export C t values to blank spread sheet (Excel). Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) -20-
21 Setting Baseline Linear View -21-
22 Setting Baseline Select AUTO CALCULATED Linear View -22-
23 Setting Threshold Log View -23-
24 Setting Threshold Threshold Line -24-
25 Setting Threshold Threshold Line C(t) -25-
26 Setting Threshold Use the Same Threshold for All PCR Arrays Threshold Line C(t) -26-
27 2 Ways to CRUNCH the Data Excel Based Templates Free! Download from Good for 2 Group Comparisons (Control + Experimental) 10 PCR Arrays per Group Web-Based Data Analysis Free! Upload Excel spreadsheet at Good for 11 Group Comparisons (Control + 10 Experimental) 255 PCR Arrays Total -27-
28 2 Ways to CRUNCH the Data Excel Based Templates Free! Download from Good for 2 Group Comparisons (Control + Experimental) 10 PCR Arrays per Group Web-Based Data Analysis Free! Upload Excel spreadsheet at Good for 11 Group Comparisons (Control + 10 Experimental) 255 PCR Arrays Total -28-
29 2 Ways to CRUNCH the Data Excel Based Templates Free! Download from Good for 2 Group Comparisons (Control + Experimental) 10 PCR Arrays per Group Web-Based Data Analysis Free! Upload Excel spreadsheet at Good for 11 Group Comparisons (Control + 10 Experimental) 255 PCR Arrays Total -29-
30 Organizing Raw C(t) values Download Excel Template from SABiosciences Web Portal or make your own. Cataloged Array Row 1 Sample Name Column A: Well Location Column B-??: Raw C(t) Values -30-
31 Organizing Raw C(t) values Download Excel Template from SABiosciences Web Portal or make your own. Cataloged Array Row 1 Sample Name Column A: Well Location Column B-??: Raw C(t) Values -31-
32 Organizing Raw C(t) values Download Excel Template from SABiosciences Web Portal or make your own. Cataloged Array Row 1 Sample Name Column A: Well Location Column B-??: Raw C(t) Values -32-
33 Organizing Raw C(t) values Download Excel Template from SABiosciences Web Portal or make your own. Cataloged Array Row 1 Sample Name Column A: Well Location Column B-??: Raw C(t) Values -33-
34 Organizing Raw C(t) values Download Excel Template from SABiosciences Web Portal or make your own. Custom Array Row 1 Sample Name Column A: Well Location Column B: Gene Name Column C-??: Raw C(t) Values -34-
35 Organizing Raw C(t) values Download Excel Template from SABiosciences Web Portal or make your own. Custom Array Row 1 Sample Name Column A: Well Location Column B: Gene Name Column C-??: Raw C(t) Values Custom Array format can be adapted for Individual PCR Assays -35-
36 Organizing Raw C(t) values Download Excel Template from SABiosciences Web Portal or make your own. LEAVE BLANK Well # UPLOAD AS CUSTOM ARRAY: CPCRDATA Custom Array Row 1 Sample Name Column A: Well Location Column B: Gene Name Custom Array format can be adapted for Individual PCR Assays Column C-??: Raw C(t) Values -36-
37 Our Experiment Control A PMA + Ionomycin 24 hours A C B Group 3 B C PMA + Ionomycin A 6 hours Group 2 B A C B Group 1 C Time 0 (resting 3 6 biological hr) replicates that will be grouped into (3 groups + control) Time 24 hours (resting) -37-
38 Our Experiment-Data Analysis Overview Control Group 1 Group 2 Group 3 A B C A B C A B C A B C 3 biological replicates that will be grouped into (3 groups + control) -38-
39 Our Experiment-Data Analysis Overview Control Group 1 Group 2 Group 3 A B C A B C A B C A B C A B C A B C A B C A B C 1 PCR Array for Each Sample (12 PCR Arrays Total) -39-
40 Our Experiment-Data Analysis Overview Control Group 1 Group 2 Group 3 A B C A B C A B C A B C A B C A B C A B C A B C C(t) C(t) GOI -C(t) HKG 1. Calculate C(t) for on each array for each GOI (Gene Of Interest) -40-
41 Our Experiment-Data Analysis Overview Control Group 1 Group 2 Group 3 A B C A B C A B C A B C A B C A B C A B C A B C C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t)+ C(t)+ C(t) 3 C(t)+ C(t)+ C(t) 3 C(t)+ C(t)+ C(t) 3 C(t)+ C(t)+ C(t) 3 1. Calculate C(t) for on each array for each GOI (Gene Of Interest) 2. Calculate Average C(t) for each gene within a Group -41-
42 Our Experiment-Data Analysis Overview Control Group 1 Group 2 Group 3 A B C A B C A B C A B C C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t)+ C(t)+ C(t) 3 C(t) = C(t) Group1 C(t) Control Group C(t)+ C(t)+ C(t) 3 C(t) = C(t) Group2 C(t) Control Group C(t)+ C(t)+ C(t) 3 C(t) = C(t) Group3 C(t) Control Group C(t)+ C(t)+ C(t) 1. Calculate C(t) for on each array for each GOI (Gene Of Interest) 2. Calculate Average C(t) for each gene within a Group 3. Calculate C(t) for each gene between Groups 3-42-
43 Our Experiment-Data Analysis Overview Control Group 1 Group 2 Group 3 A B C A B C A B C A B C C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t) C(t)+ C(t)+ C(t) 3 C(t) = C(t) Group1 C(t) Control Group C(t)+ C(t)+ C(t) 3 C(t) = C(t) Group2 C(t) Control Group C(t)+ C(t)+ C(t) 3 C(t) = C(t) Group3 C(t) Control Group C(t)+ C(t)+ C(t) 1. Calculate C(t) for on each array for each GOI (Gene Of Interest) 2. Calculate Average C(t) for each gene within a Group 3. Calculate C(t) for each gene between Groups 4. Calculate Fold Change: 2 (- Ct) 3-43-
44 Our Experiment Control A PMA + Ionomycin 24 hours A C B Group 3 B C PMA + Ionomycin A 6 hours Group 2 B A C B Group 1 C Time 0 (resting 3 6 biological hr) replicates that will be grouped into (3 groups + control) Time 24 hours (resting) -44-
45 Our Experiment PMA + Ionomycin 24 hours PMA + Ionomycin 6 hours Control (resting 6 hr) Time 24 hours (resting)
46 Topics to be Covered. Topic I: Brief Technology and Protocol Overview Monday November 15 1pm EST (Next RT 2 PCR Array Webinar) Topic II (Today): PCR Array Data Analysis Defining Baseline and Threshold Web Portal Location / Address Uploading Raw C t Data Analyzing Data & Controls Exporting Data -46-
47 Web Portal Location
48 Uploading Raw Ct Data 1. Select Array by Catalog Number 2. Select Excel File with Raw Ct Data -48-
49 Defining Experimental Design 1. Define Array Group (Control or Group X) 2. Select Housekeeping Gene -49-
50 PCR Array Reproducibility and Sample Quality 1. Check PPC 2. Check RTC 3. Check GDC -50-
51 Select Housekeeping Gene -51-
52 Analyze Results Average C t Fold Change Fold Regulation -52-
53 Visualization of Data Heat Map -53-
54 Visualization of Data Scatter Plot -54-
55 Visualization of Data Volcano Plot -55-
56 Visualization of Data Clustergram Clustergram -56-
57 Summary 1.Set machine in Absolute Quantification / or Standard Curve Mode 2.Set baseline: (ABI, Stratagene, Bio-Rad and Eppendorf*) A. Adaptive (use auto) 3.Set threshold: A. Lower 2/3 rds of amplification plot in log view B. Use same threshold for all PCR Arrays *Roche LC480 use Second Derivative Maximum 4. Export data into excel spreadsheet. Paste raw C(t) values into correct form: (sample row 1, C(t)s according to well location.) 5. Upload data to SABiosciences web portal, or download excel data analysis spreadsheets 6. Analyze Data A. Group Biological/Technical Replicates B. QC criteria (PPC, RTC, GDC) C. Focus on Stable Housekeeping Genes D. Fold Change Data E. Export Data and Publish results -57-
58 Select Up-Coming Webinars. What do I do Next? Profile the Methylation Status of Multiple Genes Simultaneously Without Bisulfite The DNA methylation profiles of promoter CpG islands are important epigenetic biomarkers to both cancer and development research. Traditional bisulfite-based methods are too tedious and time-consuming to effectively keep pace with the needed rate of biomarker discovery and validation. In this 45-minute seminar, learn about the new Methyl-Profiler DNA Methylation PCR Arrays, a faster, easier, and higher-throughput DNA methylation profiling method that combines the reliability of well-controlled restriction digestions with quantitative PCR detection NEW! Pilot Study Offers: New Epigenetic Technologies for Research: microrna, Chromatin IP, and DNA Methylation PCR Arrays Epigenetics has grabbed the attention of many researchers by providing new insights into cell differentiation and oncogenesis. These rapidly expanding studies examine how heritable factors not coded within genomic DNA sequences regulate gene expression. Such factors include DNA methylation, chromatin remodeling due to histone modification, and even, in some cases, microrna. The experimental methods designed to discover and correlate these factors with biological phenotypes have evolved. However, many still suffer from poor sensitivity and reliability, low-throughput, and unnecessary complexity. This seminar introduces advanced yet easy-to-use real-time PCR Array technologies offered by SABiosciences that analyze microrna as well as mrna expression profiles, CpG island DNA methylation, and chromatin immunoprecipitation fractions. Download the Webinar! Sign up at:
Introduction To Real Time Quantitative PCR (qpcr)
Introduction To Real Time Quantitative PCR (qpcr) SABiosciences, A QIAGEN Company www.sabiosciences.com The Seminar Topics The advantages of qpcr versus conventional PCR Work flow & applications Factors
Reprogramming, Screening and Validation of ipscs and Terminally Differentiated Cells using the qbiomarker PCR Array System
Reprogramming, Screening and Validation of ipscs and Terminally Differentiated Cells using the qbiomarker PCR Array System Outline of Webinar What are induced pluripotent Stem Cells (ips Cells or ipscs)?
Data Analysis on the ABI PRISM 7700 Sequence Detection System: Setting Baselines and Thresholds. Overview. Data Analysis Tutorial
Data Analysis on the ABI PRISM 7700 Sequence Detection System: Setting Baselines and Thresholds Overview In order for accuracy and precision to be optimal, the assay must be properly evaluated and a few
Quando si parla di PCR quantitativa si intende:
Quando si parla di PCR quantitativa si intende: A. Una PCR che produce grandi quantità di DNA B. Una PCR che emette quanti di luce C. Una PCR che quantifica il numero di molecole stampo presenti all inizio
Real-time PCR: Understanding C t
APPLICATION NOTE Real-Time PCR Real-time PCR: Understanding C t Real-time PCR, also called quantitative PCR or qpcr, can provide a simple and elegant method for determining the amount of a target sequence
qstar mirna qpcr Detection System
qstar mirna qpcr Detection System Table of Contents Table of Contents...1 Package Contents and Storage Conditions...2 For mirna cdna synthesis kit...2 For qstar mirna primer pairs...2 For qstar mirna qpcr
PreciseTM Whitepaper
Precise TM Whitepaper Introduction LIMITATIONS OF EXISTING RNA-SEQ METHODS Correctly designed gene expression studies require large numbers of samples, accurate results and low analysis costs. Analysis
Highly specific and sensitive quantitation
PRODUCT ULLETIN SYR Select Master Mix SYR Select Master Mix Highly specific and sensitive quantitation SYR Select Master Mix offers advanced performance at an affordable price. SYR Select Master Mix is
QIAGEN Rotor Gene Q: Software V2.0
QIAGEN Rotor Gene Q: Software V2.0 Instrument Setup Instructions for RT 2 Profiler PCR Arrays Preparation Before the Experiment (Presetting the Machine will save time for your run): Please make note of
QPCR Applications using Stratagene s Mx Real-Time PCR Platform
QPCR Applications using Stratagene s Mx Real-Time PCR Platform Dan Schoeffner, Ph.D Field Applications Scientist Dan.Schoeffner@Stratagene.com Tech. Services 800-894-1304 Polymerase Chain Reaction Melt
Mir-X mirna First-Strand Synthesis Kit User Manual
User Manual Mir-X mirna First-Strand Synthesis Kit User Manual United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Clontech Laboratories, Inc.
TITRATION OF raav (VG) USING QUANTITATIVE REAL TIME PCR
Page 1 of 5 Materials DNase digestion buffer [13 mm Tris-Cl, ph7,5 / 5 mm MgCl2 / 0,12 mm CaCl2] RSS plasmid ptr-uf11 SV40pA Forward primer (10µM) AGC AAT AGC ATC ACA AAT TTC ACA A SV40pA Reverse Primer
ab185916 Hi-Fi cdna Synthesis Kit
ab185916 Hi-Fi cdna Synthesis Kit Instructions for Use For cdna synthesis from various RNA samples This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 1
Real-time quantitative RT -PCR (Taqman)
Real-time quantitative RT -PCR (Taqman) Author: SC, Patti Lab, 3/03 This is performed as a 2-step reaction: 1. cdna synthesis from DNase 1-treated total RNA 2. PCR 1. cdna synthesis (Advantage RT-for-PCR
PrimePCR Assay Validation Report
Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID papillary renal cell carcinoma (translocation-associated) PRCC Human This gene
Real time and Quantitative (RTAQ) PCR. so I have an outlier and I want to see if it really is changed
Real time and Quantitative (RTAQ) PCR or.. for this audience so I have an outlier and I want to see if it really is changed Nigel Walker, Ph.D. Laboratory of Computational Biology and Risk Analysis, Environmental
User Manual/Hand book. qpcr mirna Arrays ABM catalog # MA003 (human) and MA004 (mouse)
User Manual/Hand book qpcr mirna Arrays ABM catalog # MA003 (human) and MA004 (mouse) Kit Components Cat. No. MA003...Human Whole Genome mirna qpcr Profiling Kit (-20 C) The following components are sufficient
2.500 Threshold. 2.000 1000e - 001. Threshold. Exponential phase. Cycle Number
application note Real-Time PCR: Understanding C T Real-Time PCR: Understanding C T 4.500 3.500 1000e + 001 4.000 3.000 1000e + 000 3.500 2.500 Threshold 3.000 2.000 1000e - 001 Rn 2500 Rn 1500 Rn 2000
Speed Matters - Fast ways from template to result
qpcr Symposium 2007 - Weihenstephan Speed Matters - Fast ways from template to result March 28, 2007 Dr. Thorsten Traeger Senior Scientist, Research and Development - 1 - Overview Ạgenda Fast PCR The Challenges
Essentials of Real Time PCR. About Sequence Detection Chemistries
Essentials of Real Time PCR About Real-Time PCR Assays Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected
miscript mirna PCR Array Handbook
May 2012 miscript mirna PCR Array Handbook miscript II RT Kit miscript SYBR Green PCR Kit miscript mirna PCR Arrays miscript mirna QC PCR Array For SYBR Green-based, real-time PCR profiling of micrornas
ChIP TROUBLESHOOTING TIPS
ChIP TROUBLESHOOTING TIPS Creative Diagnostics Abstract ChIP dissects the spatial and temporal dynamics of the interactions between chromatin and its associated factors CD Creative Diagnostics info@creative-
Gene Expression Assays
APPLICATION NOTE TaqMan Gene Expression Assays A mpl i fic ationef ficienc yof TaqMan Gene Expression Assays Assays tested extensively for qpcr efficiency Key factors that affect efficiency Efficiency
Validating Microarray Data Using RT 2 Real-Time PCR Products
Validating Microarray Data Using RT 2 Real-Time PCR Products Introduction: Real-time PCR monitors the amount of amplicon as the reaction occurs. Usually, the amount of product is directly related to the
RNA Extraction and Quantification, Reverse Transcription, and Real-time PCR (q-pcr)
RNA Extraction and Quantification, Reverse Transcription, and Real-time Preparation of Samples Cells: o Remove media and wash cells 2X with cold PBS. (2 ml for 6 well plate or 3 ml for 6cm plate) Keep
bitter is de pil Linos Vandekerckhove, MD, PhD
4//24 Current HIV care HIV copies/ ml plasma Viral load Welcome to the Digital droplet PCR age! bitter is de pil Linos Vandekerckhove, MD, PhD Latent HIV reservoir Time at Ghent University Hospital 2 HIV
MystiCq microrna cdna Synthesis Mix Catalog Number MIRRT Storage Temperature 20 C
microrna cdna Synthesis Mix Catalog Number MIRRT Storage Temperature 20 C Product Description The microrna cdna Synthesis Mix has been designed to easily convert micrornas into cdna templates for qpcr
Beginner s Guide to Real-Time PCR
Beginner s Guide to Real-Time PCR 02 Real-time PCR basic principles PCR or the Polymerase Chain Reaction has become the cornerstone of modern molecular biology the world over. Real-time PCR is an advanced
Tutorial for proteome data analysis using the Perseus software platform
Tutorial for proteome data analysis using the Perseus software platform Laboratory of Mass Spectrometry, LNBio, CNPEM Tutorial version 1.0, January 2014. Note: This tutorial was written based on the information
REAL TIME PCR USING SYBR GREEN
REAL TIME PCR USING SYBR GREEN 1 THE PROBLEM NEED TO QUANTITATE DIFFERENCES IN mrna EXPRESSION SMALL AMOUNTS OF mrna LASER CAPTURE SMALL AMOUNTS OF TISSUE PRIMARY CELLS PRECIOUS REAGENTS 2 THE PROBLEM
mircute mirna qpcr Detection Kit (SYBR Green)
mircute mirna qpcr Detection Kit (SYBR Green) For detection of mirna using real-time RT-PCR (SYBR Green I) www.tiangen.com QP110302 mircute mirna qpcr Detection Kit (SYBR Green) Kit Contents Cat. no. FP401
User Manual. Reference Gene Panel Mouse Probe protocol. Version 1.1 August 2014 For use in quantitative real-time PCR
User Manual Reference Gene Panel Mouse Probe protocol Version 1.1 August 2014 For use in quantitative real-time PCR Reference Gene Panel Mouse Table of contents Background 4 Assays included in the panel
qpcr Quantification Protocol Guide
qpcr Quantification Protocol Guide FOR RESEARCH USE ONLY Topics 3 Introduction 5 User-Supplied Consumables and Equipment 7 Select Template 8 Dilute qpcr Template 9 Dilute Libraries 10 Prepare Reaction
quantitative real-time PCR, grain, simplex DNA extraction: PGS0426 RT-PCR: PGS0494 & PGS0476
BioScience quantitative real-time PCR, grain, simplex DNA extraction: PGS0426 RT-PCR: PGS0494 & PGS0476 This method describes a Real-time semi-quantitative TaqMan PCR procedure for the determination of
Real-Time PCR Vs. Traditional PCR
Real-Time PCR Vs. Traditional PCR Description This tutorial will discuss the evolution of traditional PCR methods towards the use of Real-Time chemistry and instrumentation for accurate quantitation. Objectives
Mir-X mirna First-Strand Synthesis and SYBR qrt-pcr
User Manual Mir-X mirna First-Strand Synthesis and SYBR qrt-pcr User Manual United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Clontech Laboratories,
Supplemental Material. Methods
Supplemental Material Methods Measurement of lncrnas expression Total RNA was extracted from PAXgene TM tubes using the PAXgene blood RNA kit (Qiagen, Venlo, Netherlands) as described by the manufacturer.
Analysis of gene expression data. Ulf Leser and Philippe Thomas
Analysis of gene expression data Ulf Leser and Philippe Thomas This Lecture Protein synthesis Microarray Idea Technologies Applications Problems Quality control Normalization Analysis next week! Ulf Leser:
Stratagene QPCR Mouse Reference Total RNA
Stratagene QPCR Mouse Reference Total RNA Instruction Manual Catalog #750600 Revision C.0 For Research Use Only. Not for use in diagnostic procedures. 750600-12 LIMITED PRODUCT WARRANTY This warranty limits
School of Nursing. Presented by Yvette Conley, PhD
Presented by Yvette Conley, PhD What we will cover during this webcast: Briefly discuss the approaches introduced in the paper: Genome Sequencing Genome Wide Association Studies Epigenomics Gene Expression
Thermo Scientific DyNAmo cdna Synthesis Kit for qrt-pcr Technical Manual
Thermo Scientific DyNAmo cdna Synthesis Kit for qrt-pcr Technical Manual F- 470S 20 cdna synthesis reactions (20 µl each) F- 470L 100 cdna synthesis reactions (20 µl each) Table of contents 1. Description...
Quantitative Real Time PCR Protocol. Stack Lab
Quantitative Real Time PCR Protocol Stack Lab Overview Real-time quantitative polymerase chain reaction (qpcr) differs from regular PCR by including in the reaction fluorescent reporter molecules that
REAL TIME PCR SYBR GREEN
REAL TIME PCR SYBR GREEN 1 THE PROBLEM NEED TO QUANTITATE DIFFERENCES IN mrna EXPRESSION SMALL AMOUNTS OF mrna LASER CAPTURE SMALL AMOUNTS OF TISSUE PRIMARY CELLS PRECIOUS REAGENTS 2 THE PROBLEM QUANTITATION
TaqMan Fast Advanced Master Mix. Protocol
TaqMan Fast Advanced Master Mix Protocol For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. APPLIED
Gene Expression Macro Version 1.1
Gene Expression Macro Version 1.1 Instructions Rev B 1 Bio-Rad Gene Expression Macro Users Guide 2004 Bio-Rad Laboratories Table of Contents: Introduction..................................... 3 Opening
miracle mirna qpcr Instruction Manual Store at -20 Version 3.0 (Aug.2013)
miracle mirna qpcr Instruction Manual Store at -20 Version 3.0 (Aug.2013) Contents I. PRODUCT INTRODUCTION 1 A. OVERVIEW 1 B. KIT CONTENTS 1 1. REAGENT KITS 1 2. PRIMER SETS 3 3. MIRACLE ON CALL SCREEN
Introduction To Epigenetic Regulation: How Can The Epigenomics Core Services Help Your Research? Maria (Ken) Figueroa, M.D. Core Scientific Director
Introduction To Epigenetic Regulation: How Can The Epigenomics Core Services Help Your Research? Maria (Ken) Figueroa, M.D. Core Scientific Director Gene expression depends upon multiple factors Gene Transcription
Gene Expression Assay Performance Guaranteed With the TaqMan Assays QPCR Guarantee Program
WHITE PAPER TaqMan Assays QPCR Guarantee Program Gene Expression Assay Performance Guaranteed With the TaqMan Assays QPCR Guarantee Program Real-Time PCR for the Quantification of Gene Expression Real-time
SYBR Green Realtime PCR Master Mix -Plus-
Instruction manual SYBR Green Realtime PCR Master Mix -Plus- 0810 F0925K SYBR Green Realtime PCR Master Mix -Plus- Contents QPK-212T 1mLx1 QPK-212 1mLx5 Store at -20 C, protected from light [1] Introduction
PrimePCR Assay Validation Report
Gene Information Gene Name sorbin and SH3 domain containing 2 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID SORBS2 Human Arg and c-abl represent the mammalian
User Manual. CelluLyser Lysis and cdna Synthesis Kit. Version 1.4 Oct 2012 From cells to cdna in one tube
User Manual CelluLyser Lysis and cdna Synthesis Kit Version 1.4 Oct 2012 From cells to cdna in one tube CelluLyser Lysis and cdna Synthesis Kit Table of contents Introduction 4 Contents 5 Storage 5 Additionally
User Manual Mouse Endogenous Control Gene Panel
User Manual Mouse Endogenous Control Gene Panel Version 1.5 March 2008 For use in quantitative real-time PCR Mouse Endogenous Control Gene Panel Table of contents Background 4 Contents 4 Additionally required
Partek Methylation User Guide
Partek Methylation User Guide Introduction This user guide will explain the different types of workflow that can be used to analyze methylation datasets. Under the Partek Methylation workflow there are
Materials and Methods. Blocking of Globin Reverse Transcription to Enhance Human Whole Blood Gene Expression Profiling
Application Note Blocking of Globin Reverse Transcription to Enhance Human Whole Blood Gene Expression Profi ling Yasmin Beazer-Barclay, Doug Sinon, Christopher Morehouse, Mark Porter, and Mike Kuziora
FastLine cell cdna Kit
1. FastLine cell cdna Kit For high-speed preparation of first-strand cdna directly from cultured cells without RNA purification www.tiangen.com RT100701 FastLine cell cdna Kit Cat. no. KR105 Kit Contents
Genomic DNA detection assay
Genomic DNA detection assay Detection of genomic DNA by real-time PCR Contents CTRL Internal controls and gdna detection Contents Kit Contents 3 Reagents and Equipment to Be Supplied by User 3 Kit Storage
How many of you have checked out the web site on protein-dna interactions?
How many of you have checked out the web site on protein-dna interactions? Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail. Find and be ready to discuss
Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION
Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Susan Carson Heather B. Miller D.Scott Witherow ELSEVIER AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN
New Technologies for Sensitive, Low-Input RNA-Seq. Clontech Laboratories, Inc.
New Technologies for Sensitive, Low-Input RNA-Seq Clontech Laboratories, Inc. Outline Introduction Single-Cell-Capable mrna-seq Using SMART Technology SMARTer Ultra Low RNA Kit for the Fluidigm C 1 System
ONLINE SUPPLEMENTAL MATERIAL. Allele-Specific Expression of Angiotensinogen in Human Subcutaneous Adipose Tissue
ONLINE SUPPLEMENTAL MATERIAL Allele-Specific Expression of Angiotensinogen in Human Subcutaneous Adipose Tissue Sungmi Park 1, Ko-Ting Lu 1, Xuebo Liu 1, Tapan K. Chatterjee 2, Steven M. Rudich 3, Neal
Introduction to transcriptome analysis using High Throughput Sequencing technologies (HTS)
Introduction to transcriptome analysis using High Throughput Sequencing technologies (HTS) A typical RNA Seq experiment Library construction Protocol variations Fragmentation methods RNA: nebulization,
Quantitative 1-Schritt-DNA-Methylierungsanalyse aus genomischer DNA
Quantitative 1-Schritt-DNA-Methylierungsanalyse aus genomischer DNA Molekulare Diagnostik 2011 Departement Klinische Forschung Abteilung für Humangenetik Experimentelle Hämatologie DKF und Labor für Molekulare
Human Herpes Virus 4 (Epstein Barr)
Techne qpcr test Human Herpes Virus 4 (Epstein Barr) nonglycosylated membrane protein (BNRF1) gene 150 tests For general laboratory and research use only 1 Introduction to Human Herpes Virus 4 (Epstein
CompleteⅡ 1st strand cdna Synthesis Kit
Instruction Manual CompleteⅡ 1st strand cdna Synthesis Kit Catalog # GM30401, GM30402 Green Mountain Biosystems. LLC Web: www.greenmountainbio.com Tel: 800-942-1160 Sales: Sales@ greenmountainbio.com Support:
amplification tech Optical Design of CFX96 Real-Time PCR Detection System Eliminates the Requirement of a Passive Reference Dye
amplification tech note 6047 Optical Design of CFX96 Real-Time PCR Detection System Eliminates the Requirement of a Passive Reference Dye Liz Jordan and Richard Kurtz, Gene Expression Division, io-rad
Single-Cell DNA Sequencing with the C 1. Single-Cell Auto Prep System. Reveal hidden populations and genetic diversity within complex samples
DATA Sheet Single-Cell DNA Sequencing with the C 1 Single-Cell Auto Prep System Reveal hidden populations and genetic diversity within complex samples Single-cell sensitivity Discover and detect SNPs,
RealStar HBV PCR Kit 1.0 11/2012
RealStar HBV PCR Kit 1.0 11/2012 RealStar HBV PCR Kit 1.0 For research use only! (RUO) Product No.: 201003 96 rxns INS-201000-GB-02 Store at -25 C... -15 C November 2012 altona Diagnostics GmbH Mörkenstraße
All-in-One mirna qrt-pcr Reagent Kits For quantitative detection of mature mirna
All-in-One mirna qrt-pcr Reagent Kits For quantitative detection of mature mirna All-in-One TM mirna First-Strand cdna Synthesis Kit AMRT-0020 (20 RT reactions), AMRT-0060 (60 RT reactions) Used in combination
MultiQuant Software 2.0 for Targeted Protein / Peptide Quantification
MultiQuant Software 2.0 for Targeted Protein / Peptide Quantification Gold Standard for Quantitative Data Processing Because of the sensitivity, selectivity, speed and throughput at which MRM assays can
DNA Integrity Number (DIN) For the Assessment of Genomic DNA Samples in Real-Time Quantitative PCR (qpcr) Experiments
DNA Integrity Number () For the Assessment of Genomic DNA Samples in Real-Time Quantitative PCR (qpcr) Experiments Application Note Nucleic Acid Analysis Author Arunkumar Padmanaban Agilent Technologies,
A guide to setting up pay periods (including week 53) PM00104.0315/1
A guide to setting up pay periods (including week 53) PM00104.0315/1 Within this document you will find instructions for creating new pay periods and amending existing pay periods for the 2015/2016 tax
Eppendorf PCR Consumables Compatibility Guide for PCR and qpcr Cyclers
APPLICATION NOTE No. 266 I November 2012 Eppendorf PCR Consumables Compatibility Guide for PCR and qpcr Cyclers Rafal Grzeskowiak 1, Sandrine Hamels 2, Beate Riekens 1 1 Eppendorf AG, Hamburg, Germany;
Application Note. Single Cell PCR Preparation
Application Note Single Cell PCR Preparation From Automated Screening to the Molecular Analysis of Single Cells The AmpliGrid system is a highly sensitive tool for the analysis of single cells. In combination
AGILENT S BIOINFORMATICS ANALYSIS SOFTWARE
ACCELERATING PROGRESS IS IN OUR GENES AGILENT S BIOINFORMATICS ANALYSIS SOFTWARE GENESPRING GENE EXPRESSION (GX) MASS PROFILER PROFESSIONAL (MPP) PATHWAY ARCHITECT (PA) See Deeper. Reach Further. BIOINFORMATICS
Factors Influencing Multiplex Real-Time PCR
APPLICATION NOTE Multiplex Real-Time PCR Factors Influencing Multiplex Real-Time PCR Introduction Multiplex PCR is the simultaneous amplification of more than one target sequence in a single reaction [1].
Generating ABI PRISM 7700 Standard Curve Plots in a Spreadsheet Program
Generating ABI PRISM 7700 Standard Curve Plots in a Spreadsheet Program Overview The goal of this tutorial is to demonstrate the procedure through which analyzed data generated within an ABI PRISM 7700
ncounter Leukemia Fusion Gene Expression Assay Molecules That Count Product Highlights ncounter Leukemia Fusion Gene Expression Assay Overview
ncounter Leukemia Fusion Gene Expression Assay Product Highlights Simultaneous detection and quantification of 25 fusion gene isoforms and 23 additional mrnas related to leukemia Compatible with a variety
Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR
Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR Overview Genomic DNA (gdna) and plasmids containing cloned target sequences are commonly used as standards
Profiling of microrna in Blood Serum/Plasma. Guidelines for the mircury LNA TM Universal RT microrna PCR System
Profiling of microrna in Blood Serum/Plasma Guidelines for the mircury LNA TM Universal RT microrna PCR System Table of Contents 2 Introduction.....................................................................................
Scott Reierstad. Field Applications Scientist
Real-time PCR Training Scott Reierstad Field Applications Scientist Topics we ll cover Quick introduction to real-time PCR ABI-supported real-time chemistries Instrumentation Quantitation via Standard
Methods and Application Guide. Introduction to Quantitative PCR
Methods and Application Guide Introduction to Quantitative PCR Introduction to Quantitative PCR Methods and Application Guide Stratagene USA and Canada Order: 800-424-5444 x3 Technical Services: 800-894-1304
Molecular Assessment of Dried Blood Spot Quality during Development of a Novel Automated. Screening
Molecular Assessment of Dried Blood Spot Quality during Development of a Novel Automated in situ TREC qpcr Assay for SCID Screening J Bai, T Henry, J Benfer, S Berberich, T Kreman, and L DesJardin State
CABIOS. Q-RT-PCR: data analysis software for measurement of gene expression by competitive RT-PCR
CABIOS Vol. 73 no. 6 1997 Pages 587-591 Q-RT-PCR: data analysis software for measurement of gene expression by competitive RT-PCR Peter A. Doris 2, Amanda Hayward-Lester and Jon K. Hays Sr 1 Department
AffinityScript QPCR cdna Synthesis Kit
AffinityScript QPCR cdna Synthesis Kit INSTRUCTION MANUAL Catalog #600559 Revision C.01 For In Vitro Use Only 600559-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this
QuantStudio 12K Flex Real-Time PCR System. The all-in-one qpcr instrument
QuantStudio 12K Flex Real-Time PCR System The all-in-one qpcr instrument Expand the boundaries of your research Life Technologies is taking qpcr to the next level. Designed for maximum throughput, flexibility,
Next Generation Sequencing
Next Generation Sequencing Technology and applications 10/1/2015 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 1 Landmarks in DNA sequencing 1953 Discovery of DNA double helix structure 1977
Cluster Generation. Module 2: Overview
Cluster Generation Module 2: Overview Sequencing Workflow Sample Preparation Cluster Generation Sequencing Data Analysis 2 Cluster Generation 3 5 DNA (0.1-5.0 μg) Library preparation Single Cluster molecule
GSR Microarrays Project Management System
GSR Microarrays Project Management System A User s Guide GSR Microarrays Vanderbilt University MRBIII, Room 9274 465 21 st Avenue South Nashville, TN 37232 microarray@vanderbilt.edu (615) 936-3003 www.gsr.vanderbilt.edu
TaqMan Genotyper Software v1.0.1 TaqMan Genotyping Data Analysis Software
TaqMan Genotyper Software v1.0.1 TaqMan Genotyping Data Analysis Software March 2011 Product Overview TaqMan Genotyper Software TaqMan Genotyper Software Standalone data analysis software for Applied Biosystems
Real-time qpcr Assay Design Software www.qpcrdesign.com
Real-time qpcr Assay Design Software www.qpcrdesign.com Your Blueprint For Success Informational Guide 2199 South McDowell Blvd Petaluma, CA 94954-6904 USA 1.800.GENOME.1(436.6631) 1.415.883.8400 1.415.883.8488
Description: Molecular Biology Services and DNA Sequencing
Description: Molecular Biology s and DNA Sequencing DNA Sequencing s Single Pass Sequencing Sequence data only, for plasmids or PCR products Plasmid DNA or PCR products Plasmid DNA: 20 100 ng/μl PCR Product:
Hepatitis B Virus. genesig Advanced Kit. DNA testing. Everything... Everyone... Everywhere... Core Protein Region. 150 tests.
TM Primerdesign Ltd TM Primerdesign Ltd Hepatitis B Virus Core Protein Region genesig Advanced Kit 150 tests DNA testing Everything... Everyone... Everywhere... For general laboratory and research use
PNA BRAF Mutation Detection Kit
- PNA BRAF Mutation Detection Kit Catalog Number KA2102 50 tests/kit Version: 01 Intended for research use only www.abnova.com Introduction and Background Intended use The PNA BRAF Mutation Detection Kit
Improving qpcr Reliability: Automated RNA Normalization and Plate Setup on PIPETMAX
Improving qpcr Reliability: Automated RNA Normalization and Plate Setup on PIPETMAX Application Note TRANS915 Normalization of RNA concentrations before reverse transcription reduces error in qpcr experiments
Absolute Quantification Getting Started Guide
5 cdna Reverse Primer Oligo d(t) or random hexamer Synthesis of 1st cdna strand 3 5 cdna Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide Introduction
Multiplex your most important
Multiplex your most important genetic assays on one platform GenomeLab GeXP Genetic Analysis System Blood Banking Capillary Electrophoresis Centrifugation Flow Cytometry Genomics Lab Automation Lab Tools
Dengue Virus subtypes 1,2 3 and 4. genesig Standard Kit. DNA testing. Everything... Everyone... Everywhere... 3 Untranslated Region (3 UTR) 150 tests
TM Primerdesign Ltd TM Primerdesign Ltd Dengue Virus subtypes 1,2 3 and 4 3 Untranslated Region (3 UTR) genesig Standard Kit 150 tests DNA testing Everything... Everyone... Everywhere... For general laboratory
DyNAmo cdna Synthesis Kit for qrt-pcr
DyNAmo cdna Synthesis Kit for qrt-pcr Instruction manual F- 470S Sufficient for 20 cdna synthesis reactions (20 µl each) F- 470L Sufficient for 100 cdna synthesis reactions (20 µl each) Description...
Appendix 2 Molecular Biology Core Curriculum. Websites and Other Resources
Appendix 2 Molecular Biology Core Curriculum Websites and Other Resources Chapter 1 - The Molecular Basis of Cancer 1. Inside Cancer http://www.insidecancer.org/ From the Dolan DNA Learning Center Cold
Plexor Systems Instrument Setup and Data Analysis for the Applied Biosystems 7300 and 7500 Real-Time PCR Systems
Technical Manual Plexor Systems Instrument Setup and Data Analysis for the Applied Biosystems 7300 and 7500 Real-Time PCR Systems INSTRUCTIONS FOR USE OF PRODUCTS A4011, A4021, A4031, A4041, A4051 AND