how to measure gene xpression

Question 1

realttimePCR ~ I have a graph of number of PCR cycles and relative fluorescence on y-axis.
If my curve is shifted to the right this means
A expression of gene low
B expression of gene high
C doesnt indicate expression of gene

Answer 1

option A - if curve shifts to right then there is low expression of that gene (relative to another curve to the left) as it took more cycles to cross that threshold so that tells me there wasn’t alot to amplify in the first place

qPCR yaay

Question 2

A scientist is using the CRIPSR/Cas9 system to inactivate a particular target gene.  What is the component of the CRISPR/Cas9 system that directs it to the correct target?
A A single stranded DNA molecule.
B A single stranded RNA molecule.
C A zinc finger nuclease.
D The Cas9 protein.
E The Cre protein.

Answer 2

option B

A guide RNA that has a region complementary to one strand of the target DNA sequence.
It guides a nuclease (Cas9 most common) to a DNA target.
The protospacer adjacent motif (PAM sequence) – NGG for Cas9 – is needed on the complementary strand immediately after the target site

Question 3

what is the key tool used to detect a mRNA molecule?

Answer 3

uses PROBES to detect a specific mRNA molecule and it will HYBDRISE with that specific mRNA molecule!

Question 4

what does northern blotting detect?

Answer 4

detects RNA molecules using gel electrophoresis onto a nylon membrane and then treated with a probe
and then expose to X-ray to see the bands

Question 5

what is a normalisation control?

Answer 5

its a housekeeping gene and always used in any blotting or PCR to make sure that that the quantity and quality of RNA product is good// DETECTS ANY VARIATION IN THE LOAD OF EACH LANE

comparison point as it should have same expression throughout

Question 6

what is the prbe used in western blo?t

Answer 6

western blot looks for specific proteins and does this by separating protein by SDS page so will use a ANTIBODY AS probe

Question 7

how does south and north blot differ?

Answer 7

south - DNA, will denature the DNA in sodium hydroxide before transfering to the membrane. but same probe treatment is used

north - RNA

Question 8

what is a negative control in PCR? (rt-PCR)

Answer 8

PCR is prone to contamination so if we do a reaction with no reverse transcriptase activity

then there is nothing for the primers to bind to and so nothing should be amplified = no bands should show on the gel elec

Question 9

why does rtPCR have limited dynamic range// why is 40 cycles our plateau point

Answer 9

because we have run out of dNTP/primers/reagents

because the 2 PCR strands are annealing to each other instead of the primers