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Real Time Quantitative PCR

Theory, experimental design and data analysis

 (3.5 credits)

November 29th-December 19th 2022

Registration deadline November 11th, 2022.

Schedule

Lecture 1. Basics of PCR and qPCR

Tuesday, November 29. 9:00am – 12:00pm

Zoom link
Meeting ID: 613 3487 5245
Passcode: 573726

Feedback form

Lecture 2. qPCR

Wednesday, November 30. 9:00am – 12:00pm

Zoom link
Meeting ID: 623 8593 4206
Passcode: 414710

Feedback form

Lecture 3. Affymetrix, Transcriptomics and tools useful for qPCR

Thursday, December 1. 9:00am – 12:00pm

Zoom link
Meeting ID: 649 2625 1064
Passcode: 236080

Feedback form

Seminar 1. Ins and outs of a qPCR experiment design

Friday, December 2. 9:00am – 12:00pm

Zoom link
Meeting ID: 655 6780 5579
Passcode: 240600

Groups for practical part

Feedback form

Seminar 2. Experiment design in groups

Monday, December 5. 1:15 – 4:15pm

Zoom link
Meeting ID: 621 5403 8064
Passcode: 961092

Feedback form

Group Homework 1.

Tuesday December 6, whole day

Location:-

Each group will design primers required for their experiment.

Group Homework 2.

Wednesday-Thursday, December 7-8, whole day

Location:-

Each group will prepare step by step protocol for analysis of their qPCR data

Seminar 3. Discussion of plate layout, data analysis and presentation.

Friday, December 9. 1:15 – 4:15pm

Zoom link
Meeting ID: 658 1936 7214
Passcode: 069907 

qPCR lab work.

December 12, – December 14, whole day.

Location:Uppsala BioCenter, 3d floor, C342

Each group will pipette their qPCR experiment.

Group Homework 3.

December 15 – 16, 2022, whole day

Location:-

Each group will analyze their qPCR data and prepare presentation for the final seminar

Seminar 4. Reports.

Monday, December 19. 9:00am – 12:00pm

Zoom link
Meeting ID: 659 5073 6222
Passcode: 053745

​​​Useful stuff

Manual for run setup and data analysis

MIQE guidelines

How to find linear regression in Excel

Efficiency calculation

Short guide for qPCR data analysis

Use of betaine for GC-rich templates

Use of DMSO for GC-rich templates

Molecular Biology of Cell (NCBI bookshelf)

Intro into logarithms

 

Selecting reference genes

GENEVESTIGATOR (make sure JAVA is of the latest version and is enabled in your internet browser)

 

Primer design

Guidelines for primer design

Primer3Plus

Primer-BLAST

Software for data analysis (for Windows only!)

CFX Manager, to install:

  1. download the file

  2. unzip CFX Manager 3.1.zip

  3. open the folder CFX_Manager

  4. run the Setup file

 

Short manual for qPCR machine software:

  CFX short manual    

 

 

 

SYBR Green kit

ThermoFisher manual

 

 

 

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

Syllabus approved: 2022.10

 

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

  1. (2009). The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry, 55(4), 611–622.

  2. http://statistics.gene-quantification.info/

 

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