Lab 5

Question 1

hsp-16

Answer 1

chaperone

Question 2

mcherry

Answer 2

red fluor. protein

Question 3

explain how mcherry is expressed

Answer 3

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

Question 4

what is exp 2b studying

Answer 4

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)

Question 5

what is exp 2c studying

Answer 5

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

Question 6

what is exp 3 studying

Answer 6

does disruption of mito function activate cellular stress response? we will use reporter worm to see if there’s activation of heat shock response

Question 7

why are we inclduding mcherry in experimeent?

Answer 7

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?

Question 8

what plates are we using for exp 3 and what are the controls

Answer 8

(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)

Question 9

goal of pcr

Answer 9

amplify a specific dna sequence

Question 10

How can you detect or measure gene expression?

Answer 10

● 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

Question 11

cDNA

Answer 11

Copy or complementary dna. reverse transcriptase converts mRNA to cDNA during qpcr.

Question 12

qPCR

Answer 12

allows determination of the relative concentration of mrna in a sample. mrna is converted to cdna. then cdna is amplified

Question 13

taq polymerase

Answer 13

amplifies cdna in pcr

Question 14

Pcr

Answer 14

Amplifies cDNA made from qpcr

Question 15

when quencher and reporter dye in probe is close together, there is/isn’t a signal

Answer 15

isn’t

Question 16

how do we get a signal from probe in RT QPCR

Answer 16

probe is displaced by primer (that’s on CDNA), probe breaks so quencher and reported are separated

Question 17

Ct (threshold cycle)

Answer 17

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.

Question 18

how are we breaking worms open

Answer 18

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

Question 19

housekeeping gene

Answer 19

gene that’s expression doesn’t change under our conditions

Question 20

why are we using a housekeeping gene (cdc42)

Answer 20

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

Question 21

Why do we want to know relative concentration of mrna is the cell?

Answer 21

so we can observe changes in gene expression for proteostasis genes (increased gene expression=more relative mrna concentration)

Question 22

DNAase is used to

Answer 22

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

Question 23

we’re testing longevity to see

Answer 23

how gene knockdown affected longevity in the worms