3 replies to this topic

[molecular_medicine]

Dear All,

I am running an hypoxia experiment and use cobalt chloride(300uM) to mimic hypoxia. At different time points I collect rna from my cells and perform reverse transcription and use the cDNA for my reat time PCR. Out of the four genes of my interest, HIF-1A is one among them. When compared to the normoxic controls, HIF-1A expression is very low!! And all the 3 other genes are known to be upregulated during hypoxia and 2 among them are significantly upregulated whist 2 genes including HIF-1A is not!!!

Can you please suggest why this might be the case. The concentration of cobalt chloride is known to induce hypoxia in different cell lines.. pleeeeease help!!!

[molgen]

And when not in hypoxia, how dose the HIF-1A express in relation to the others?
According to the literature, what is it supposed to do (up or down regulate)?

[molecular_medicine]

molgen, on Apr 5 2009, 03:09 AM, said:

And when not in hypoxia, how dose the HIF-1A express in relation to the others?
According to the literature, what is it supposed to do (up or down regulate)?

During normoxia, HIF-1A expression should be unchanged wheres as it is unchanged for few hours but then it went up high around 7 hours... I am sure I havent chnaged the vials.... Please help!!

[Dr Teeth]

molecular_medicine, on Apr 5 2009, 02:23 PM, said:

molgen, on Apr 5 2009, 03:09 AM, said:

And when not in hypoxia, how dose the HIF-1A express in relation to the others?
According to the literature, what is it supposed to do (up or down regulate)?

During normoxia, HIF-1A expression should be unchanged wheres as it is unchanged for few hours but then it went up high around 7 hours... I am sure I havent chnaged the vials.... Please help!!

Are you looking at HIF-1a protein or mRNA? Since the protein level sees the most change as HIF-1a is constitutively degraded in the absence of hypoxia. Also, are you looking at other targets known to be upregulated with addition of CoCl2 like VEGF? Using other targets could help you identify whether your CoCl2 is working. Finally, what concentration of CoCl2 are you using?