On Day 1 and 2, we performed the analysis of an RNA-sequencing dataset. These data showed that the mutants displayed an alteration in the expression of genes involved in iron homeostasis regulatory processes. Given that such transcriptomic analyses sometimes retrieve false positives, it is key to validate the differential expression of interesting candidate genes using another technique. For this purpose, molecular biology labs typically perform RT-qPCR (= quantitative PCR on cDNA obtained by reverse transcription of total RNAs) analysis of candidate genes’ expression. These analyses are usually performed on RNAs extracted from samples from a fully independent experiment. The goal of the second part of these practicals (Day 2 to Day 4) will be to use RT-qPCR in order to quantify the expression levels of candidate genes in the mutant used for the RNA-seq experiment. However, given that roots are small, RNA extraction would require a large number of plants to sample. Therefore, we will perform the RNA extraction from the whole plants.
The goal of these practicals will thus be to quantify the transcript levels of selected candidate genes in frd3 mutants and to compare them with those in wild-type plants.
For this, we will perform the following steps :
The goal of today’s work will be to perform an RNA extraction from plants grown in Petri dishes and to quantify these RNAs.
Two weeks ago, seeds from two different Arabidopsis thaliana genotypes were sterilized and sowed, three days later, on Petri dishes : i. a mutant of interest and ii. wild-type plants, which will be used references. These plants were grown under 8 h short day photoperiods, 70% humidity, and a light intensity of 100 µmol/m-2/s-1.
To determine whether the mutant of interest is altered in iron homeostasis, we will perform RT-qPCR quantification of known metal homeostasis genes in order to confirm that it is indeed the Fe-homeostasis pathway that is altered in the mutant that was analyzed using the RNA-seq analysis.
The protocol that we will carry out will take 3 days and consist of 7 main steps (Steps 1-4 = Friday 17/11; Steps 5-6 = Monday 20/11; Step 7 = Wednesday 22/11):
If you want extra information for steps 1 to 3, the full manual of the RNA extraction kit is available HERE