The relevant material will be uploaded during the course
Lecture 1.
Basics of PCR and qPCR
Tuesday, April 2. 9:00 am – 12:00 pm
Location: Uppsala BioCenter, Room C-216
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Lecture 2.
qPCR normalization, reference genes, MIQE guidelines, melt curve
Wednesday, April 3. 9:00 am – 12:00 pm
Location: Ullls Hus Lecture Hall U (C-250E207)
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Lecture 3.
Affymetrix, Transcriptomics, Tools useful for qPCR
Thursday, April 4. 9:00 am – 12:00 pm
Location: Uppsala BioCenter. Room C-216
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Seminar 1.
The ins and outs of a qPCR experiment design
Friday, April 5. 9:00 am – 12:00 pm
Location: Uppsala BioCenter. Room C-213
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Seminar 2.
Experiment design in groups for an absolute and relative qPCR
Monday, April 8. 9:00 am – 12:00 pm
Location: Ulls Hus Lecture hall P, C-250B203
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Seminar 3.
Plate layout design revision. Data analysis. qPCR data publication
Friday, April 12. 9:00 am – 12:00 pm
Location: Uppsala BioCenter. Room C-216
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qPCR lab work
Pipetting qPCR, starting the program
Monday-Friday, April 15-19.
Location: Uppsala BioCenter
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Seminar 4.
Final report
Friday, April 26. 14:00 – 16:00 pm
Location: Uppsala BioCenter. Room C-212
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Useful stuff
Manual for run setup and data analysis
How to find linear regression in Excel
Short guide for qPCR data analysis
Excel template for serial dilution calculations
Use of betaine for GC-rich templates
Use of DMSO for GC-rich templates
Molecular Biology of Cell (NCBI bookshelf)
Selecting reference genes
GENEVESTIGATOR (make sure JAVA is of the latest version and is enabled in your internet browser. Alas! Now applicable only for humans, mouse and rat)
Primer design
Software for data analysis (for Windows only!)
CFX Manager, to install:
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download the file
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unzip CFX Manager 3.1.zip
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open the folder CFX_Manager
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run the Setup file
CFX Maestro,to install:
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download the file
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unzip the folder
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run the CFXMaestroSetup
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allow to install the updates
Short manual for qPCR machine software:
Demo data:
Demo of absolute and relative qPCR runs
SYBR Green kit
LOD/LOQ calculation
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tutorial video by Berbagi Pengetahuan
Course Syllabus
All lectures are open, registration is required only to attend the practical part of the course
Language: English
Subject: Biology
Marking scale: Pass / Failed
Prerequisites
The course is intended for PhD students within the SLU Graduate School Organism Biology, but is open for all interested SLU PhD students. If space allows, other SLU staff and PhD students from other universities are also welcome to apply. Lectures and seminars will be open for all SLU staff and PhD students from other universities, practical part of the course will be available only for registered attendees.
Objective
The course aims to equip students with comprehensive knowledge of applicability of qPCR methods, train relevant technical skills and introduce currently available tools. After the course students will be able to decide whether qPCR is the best method to address a given problem, design a qPCR experiment following MIQE guidelines for scientific publications of qPCR data, analyze the data and write it up for a publication.
Content
The course includes a strong theoretical background in qPCR, practically-oriented modules on the design of qPCR experiments, the analysis and presentation of qPCR data. Students will have an opportunity to learn about practical application of the qPCR method in the context of other existing techniques (NGS, Microarrays etc). The course also includes a practical part, during which students will design their own experiments, design and order primers, prepare templates, perform qPCR and analyze the data. Teaching will be carried out in the form of lectures and seminars combined with group works as well as lab work at Uppsala BioCenter. Additionally students will be asked to perform several homework tasks in groups.
Students will be encouraged to design and perform experiments based on their own project, however it will be also possible to perform an experiment using material provided by the course.
Literature
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(2009). The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry, 55(4), 611–622.
Examination
A student should attend at least 80% of teaching sessions and complete the practical and ALL homework tasks. Students will be asked to work in groups of 2-3 people. For examination, each group will make a powerpoint presentation presenting the results of their data analysis as it would be done for a publication, summarizing the principles behind the experiment layout and their conclusions. These presentations will be discussed by all course participants at the final seminar.
Course Organizers:
Alyona Minina, E-mail: alena.minina@slu.se
Katarina Landberg, E-mail: Katarina.Landberg@slu.se
Lecturers
Alyona Minina, Department of Molecular Sciences, SLU
Additional information
The course is organized by Alyona Minina (Department of Molecular Sciences, NJ faculty) on behalf of the SLU Organism Biology research school. Most of the course activities will take place at Uppsala BioCenter, Ultuna campus, SLU. Lectures and seminars might be moved to Zoom in case of unforeseen lockdowns. For the practical part students will have to attend Uppsal BioCenter in small groups. The course program is designed for maximum 15 students per course occasion.
Responsible department
Department of Plant Biology, BioCenter, SLU