Hypoxia is a well$-$documented microenvironmental factor contributing to drug resistance in cancer cells by upregulating various genes, including hypoxia$-$inducible factor l$-$alpha $($HIF$-1$a$).$ HIF$-$l $$ activates survival pathways, making cancer cells less responsive to chemotherapy. To explore potential solutions, a novel drug candidate, GBE$309,$ designed to counteract hypoxia$-$mediated resistance, is being tested.

In this experiment, you will assess the impact of GBE$309$ on HF$-$la expression in cancer cells cultured under normal and hypoxic conditions using qPCR$.$ The $q$ PCR data, including C$($t$)$ values, are presented in Tables $1$ and $2.$

Table $1.$ C$($t$)$ values of HIF$-$l $$ and GAPDH genes under normal $($normoxia$)$ conditions. Please note that your data includes both technical $($Ct$1,2,3)$ and biological replicates.

$\backslash$table$[[$Normoxia$,],[,$HIF$-$la$,$GAPDH,$],[$Ct$1,$Ct$2,$Ct$3,$Ct$1,$Ct$2,$Ct$3],[19\mathrm{.}2,17\mathrm{.}63,18\mathrm{.}57,15\mathrm{.}90,16\mathrm{.}09,15\mathrm{.}89],[17\mathrm{.}89,18\mathrm{.}49,18\mathrm{.}56,16\mathrm{.}08,16\mathrm{.}3,16\mathrm{.}13],[17\mathrm{.}29,17\mathrm{.}82,17\mathrm{.}63,16\mathrm{.}10,16\mathrm{.}16,16\mathrm{.}05]]$

Table $2.$ C$($t$)$ values of HIF$-$l $$ and GAPDH genes under hypoxic conditions, with and without Drug GBE$309.$ Please note that your data includes both technical $($Ct$1,2,3)$ and biological replicates.

$\backslash$table$[[$Hypoxia$,$Hypoxia $+$ Drug GBE$309,,,],[$HIF$-$la$,$GAPDH,,HIF$-$la$,$GAPDH$],[$Ct$1,$Ct$2,$Ct$3,$Ct$1,$Ct$2,$Ct$3,$Ctl$,$Ct$2,$Ct$3,$Ct$1,$Ct$2],[$Ct$3,,,,,,,,,,],[24\mathrm{.}65,25\mathrm{.}00,23\mathrm{.}47,24\mathrm{.}95,25\mathrm{.}23,25\mathrm{.}19,23\mathrm{.}6,22\mathrm{.}85,23\mathrm{.}32,21\mathrm{.}96,22\mathrm{.}07],[25\mathrm{.}02,25\mathrm{.}50,24\mathrm{.}76,25\mathrm{.}70,25\mathrm{.}44,25\mathrm{.}90,22\mathrm{.}92,23\mathrm{.}12,23\mathrm{.}00,22\mathrm{.}09,22\mathrm{.}64],[24\mathrm{.}75,24\mathrm{.}2,23\mathrm{.}77,24\mathrm{.}50,25\mathrm{.}46,25\mathrm{.}37,23\mathrm{.}4,23\mathrm{.}67,22\mathrm{.}89,22\mathrm{.}33,21\mathrm{.}67],[22\mathrm{.}14,,,,,,,,,,]]$

You also ran a pure product of $10$ pg cDNA for each amplicon $($HIF$-1$ and GAPDH separately$).$ You did serial dilutions and obtained the following C$($t$)$ results:

Table $3.$ C$($t$)$ values of the pure product of $10$ pg HIF$-$la and GA$.$PDH and their dilutions.

$\backslash$table$[[$Primers$,],[$Starting $10$ pg: $1$X$,$HIF$-1$a$,$GAPDH$],[1x$ dilution,$0\mathrm{.}04,0\mathrm{.}87],[10x$ dilution,$4\mathrm{.}5,4\mathrm{.}31],[100$X dilution,$7\mathrm{.}8,7\mathrm{.}76],[1000x$ dilution,$11\mathrm{.}9,11\mathrm{.}21]]$

Generate a standard curve for both primer sets used in this experiment. Show the curves for each. Calculate the efficiencies for each primer set $($HIF$-$l $$ and the housekeaping gene from Table $3).(20$ points$)$

Apply the Livak method and analyse the result by drawing a column graph that shows the fold change of HIF$-1$a$.$ Please note that, to evaluate these effects accurately, normoxia should be used as the control condition, and GAPDH should be used as the housekeeping gene for normalization. Show all the calculations over excel formulas$/$tables$.$ Apply appropriate t$-$test statistics, explain your test and show the p$-$value and error bars. $(35$ points$)$

Apply the Pfaffl method and analyse the result by drawing a column graph that shows the fold change HIF$-1$a$.$ Please note that, to evaluate these effects accurately, normoxia should be used as the control condition, and GAPDH should be used as the housekeeping gene for normalization. Show all the calculations over excel formulas$/$tables$.$ Apply appropriate t$-$test statistics, explain your test, and show the p$-$value and error bars. $(35$ points$)$

Evaluate the effect of the GBE$309$ drug on resistance under hypoxic conditions. $(10$ points$)$