Genetic and molecular approaches: assessing gene expression and genetic interactions

Question 1

What do in situ hybridisation and immunostaining techniques detect and what does this allow us to figure?

Answer 1

In situ hybridisation and immunostaining are techniques that detect the distribution in tissues of either mRNA or proteins
-This allows us to figure out what tissues are expressing particular genes

Question 2

What are the steps involved in immunohistochemistry?

Answer 2

• This method detects distribution of a protein within a tissue
• Primary antibody will bind to the protein
• A secondary antibody will be used which is directed to the primary antibody
• The secondary antibody is attached to an alkaline phosphatase which again undergoes a precipitation reaction giving a coloured precipitate.

Question 3

What does subtractive hybridisation allow?

Answer 3

-Allows us to identify differences in gene expression between regions of the embryo or between normal and mutant embryos

Question 4

What are the steps involved in subtractive hybridisation?

Answer 4

• Isolate the mRNA from the 2 samples
• We then retro transcribe the mRNA into cDNA and modify one sample
• Then we hybridise the 2 samples
• After that we remove the genes common to both samples
• Finally we isolate the remaining genes

Question 5

What does the microarray method allow us to detect?

Answer 5

-This method allows us to detect differences in levels of expression

Question 6

What are the steps involved in microarrays?

Answer 6

• We start with 2 samples and we isolate mRNA from both samples
• We then convert the mRNA to cDNA by reverse transcription
• Then we label the samples. One with a red fluorescence tag and one with a green fluorescence tag
• After that we combine both the targets and we use them as a probe to hybridise to a microarray
  
  • Microarray is a slide with many wells with each well containing a particular genome
• If the gene in the well is presented in our probe, the probe will bind to it and emit fluorescence.

Question 7

Steps involved in RNAseq

Answer 7

• Start with two samples
• Then we isolate RNAs
• Then generate cDNA, fragment, size select, add linkers
• Then we sequence all those fragments
• Once we get all the reads, we can assemble the exome or transcriptome of each sample.
• Now we can compare the levels of each sequence and compare levels of expression.