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Real Time Quantitative PCR

Theory, experimental design and data analysis

 (3.5 credits)

Course #P000008 (The Occasion code/Tillfälleskod:F0032)

 

Spring 2024

Registration deadline is March 25.

Course schedule and material

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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Visit of the NGS facility at SciLife Lab

NGS presentation and lab tour

Tuesday, April 9. 9:25-12:00

Location: SciLifeLab Uppsala, Entrance C11, BMC, Husargatan 3, 752 37 Uppsala, Sweden

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Group Homework 1

Experiment design in groups

Tuesday- Thursday, April 9-11

Location: Home

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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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Group Homework 2

Analysis of the obtained qPCR data, preparation of the presentation

Monday-Thursday, April 22-25.

Location: Home

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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

MIQE guidelines

How to find linear regression in Excel

Efficiency calculation

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)

Intro into logarithms

 

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

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

CFX Maestro,to install:

  1. download the file

  2. unzip the folder

  3. run the CFXMaestroSetup

  4. allow to install the updates

 

Short manual for qPCR machine software:

  CFX short manual    

 

Demo data:

Demo of absolute and relative qPCR runs

 

 

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

 

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

 

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