Lab 5 Flashcards
hsp-16
chaperone
mcherry
red fluor. protein
explain how mcherry is expressed
hsp-16, a chaperone, is bound to promoter of mcherry. the promoter is bound hsf1. so when heat shock response is activated, hsf1 stops binding to chaperones and binds to the promoter, activating expression
what is exp 2b studying
is polyq toxicity? does gene knockdwon affect polyq toxicity? we will use glp 1 wt and glp1 q35 worms on l4440 and l4440 goi plates (4 conditions)
what is exp 2c studying
how does gene expression of polyq35 alter expresison of proteostasis genes? how does gene knockdown alter expression of proteostasis genes?
we will use glp 1 wt and glp1 q35 worms on l4440 and l4440 goi plates (4 conditions)
we will do longevity counts to see how goi knockdown has effected longevity in wt and q35 worms
we will measure gene expression of proteostasis genes via reverse transcriptase qpcr
what is exp 3 studying
does disruption of mito function activate cellular stress response? we will use reporter worm to see if there’s activation of heat shock response
why are we inclduding mcherry in experimeent?
we’re activating it to see if HSR was activated. in other words, did disfunction of mito cause it to “talk” with nucleus and activate HSR?
what plates are we using for exp 3 and what are the controls
(L44440 + HS) L4440 W/ HS (heat shock). this is pos control
(L4440 -GOI -HS) L4440 W/ GOI knockdown, but no HS
(L4440 -HS) L4440 (control. No HS or GOI knockdown)
goal of pcr
amplify a specific dna sequence
How can you detect or measure gene expression?
● Transcription (DNA to mRNA) to see which genes are transcribed
○ mRNA from lysed worms
● Translation (mRNA to protein): for example, some mrna are held back from being translated
● Protein Stability (half-life): some proteins may be degraded when cell is under stress
○ Western blot (examine proteins)
● Protein Post-translational modifications
○ phosphorylation: one way cell alters protein activity
cDNA
Copy or complementary dna. reverse transcriptase converts mRNA to cDNA during qpcr.
qPCR
allows determination of the relative concentration of mrna in a sample. mrna is converted to cdna. then cdna is amplified
taq polymerase
amplifies cdna in pcr
Pcr
Amplifies cDNA made from qpcr
when quencher and reporter dye in probe is close together, there is/isn’t a signal
isn’t
how do we get a signal from probe in RT QPCR
probe is displaced by primer (that’s on CDNA), probe breaks so quencher and reported are separated
Ct (threshold cycle)
the cycle number at which the fluor. signal of the reaction crosses the threshold. it’s used to find initial cdna copy number(and thus mrna bc dna is converted to mrna- see slides 25/26) bc the higher the ct value, the less initial cdna strands and vice versa.
how are we breaking worms open
putting the tubes made last week in liquid nitrogen for a minute. then put in warm water bath for a minute. the ice crystals that form and this shears worm open. go back and forth 8 times. the selected genes are in genomic dna, so we need to treat worms with dnase to destroy genomic dna bc we just want to do pcvr with the selected genes, not everything
housekeeping gene
gene that’s expression doesn’t change under our conditions
why are we using a housekeeping gene (cdc42)
bc we’re comparing relative amount of mrna extracted from worms. but what if initial amount of mrna in one sample is more than other? than it will look like this sample made more mrna? so we use cdc42 because this expression won’t change.
The Cdc-42 probe is labeled with Vic (excitation 526 and emission 543). The various probes are labeled with Fam (excitation 493 and emission 517). This allows for the detection of 2 probes within the same tube.
Our calculations next week will take into account the amount of Gene X relative to Cdc42; then we will determine the relative amount compared to the control
Why do we want to know relative concentration of mrna is the cell?
so we can observe changes in gene expression for proteostasis genes (increased gene expression=more relative mrna concentration)
DNAase is used to
degrade genomic dna. any dna in worms can be amplified during pcr so we degrade rest of dna so we can just amplify sequence of interest
we’re testing longevity to see
how gene knockdown affected longevity in the worms
