Homework

Part 1. Make a thorough plan for the demo experiments

Detection of Autophagy-related genes (ATG) expression in plants

3

2024

Arabidopsis (Arabidopsis thaliana) plant in pot

Arabidopsis (Arabidopsis thaliana) seedlings grown on a Petri plate

Autophagy

4

2024

• It is the major catabolic pathway in eukaryotes

( Greek for self-eating)

• One of the key players in plant stress tolerance and pathogen interaction

5

2024

Autophagy sequesters superfluous or damaged content from the cell cytoplasm into vesicles called autophagosomes and delivers it to the lytic compartment for degradation (lytic vacuole in plants)

6

2024

Autophagy related gene 8 (ATG8) codes an ATG8 protein, which is an integral component of membrane vesicles formed by autophagy

Phagophore

Autophagosome

Autophagic body

PAS

GFP-ATG8

Arabidopsis has 9 orthologs of ATG8�ATG8a, ATG8b, ATG8… until ATG8i

7

2024

Expression data for ATG8a-i from transcriptomics experiments for plants grown under normal conditions

High expression

Low expression

For now, we want to focus on ATG8E, ATG8F and ATG8I

Compare expression of ATG8E, ATG8F and ATG8I in Arabidopsis plants of the wild-type and mutant backgrounds

• Arabidopsis seedlings(young plants) were grown under standard conditions for 7 days

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• On the day 8, seedlings of each genotype were pooled together and sampled

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• Total RNA was extracted from the seedlings and RT was performed

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• Please use qPCR to address two questions (please treat these two questions as independent experiments):
1. How much does expression of each gene (ATG8E, ATG8F and ATG8I) differ between WT and mutant plants
2. Which of ATG8 genes is expressed at highest amount and in which background (WT or mutant?)

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8

2024

Work in groups

• Design detailed layout for your qPCR experiments:
• Absolute qPCR for ATG8s
• Relative qPCR for ATG8s
• Your own experiment

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• The layout should include:
• Plates layouts
• Calculations for Master mixes and serial dilutions (NB! Dilute each cDNA 25x, total reaction V = 15 ul, Template V/reaction = 5 ul, have 3 technical replicates for each reaction)
• Plan how each component will be pipetted
• PCR program
• Plan for data analysis

Template preparation for relative/absolute qPCRs for ATG8s

• Total RNA has been extracted. RIN for all samples was >8

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• RT has been performed using first strand Maxima kit and its default conditions (1000ng of each RNA/RT reaction were used)

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• You will receive 20 ul of cDNA for WT and mutant

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• You will receive 30 ul of three plasmids containing ATG8E, ATG8F and ATG8I. Each plasmid will be at concentration 25 ng/ul:
• pGGC ATG8 E plasmid 3057 bp
• pGGC ATG8 F plasmid 3057 bp
• pGGC ATG8 I plasmid 3039 bp

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Your primers

GOI

Reference genes taken from�doi: 10.1104/pp.105.063743

Please use Primer –Blast to check primer specificity

In the CFX software

1. Create and save the PCR program for your planned qPCR
2. Create and save annotated plates layout

Part 2. Make a thorough plan for your hypothetical experiment

Please prepare the following information for your final presentation

• Pick one of the experiments you planned for the Seminar 1
• Plan a suitable sampling strategy
• Look up how you will extract nucleic acids and check their quality
• Look up what RT kit might be recommended for your use (if you need any)
• Look up if another qPCR kit would be better for your case
• Design/find and verify in silico primers
• Plan plate layout
• Calculate MM and dilutions of template

Aim to at least try obtaining this information to discuss at the seminar 3