I did RT-PCR using SYBR Green and used 2 ul of cDNA per sample to investigate the effect of LPS in primary osteoclast culture.
I loaded the same amount of RNA to make cDNA (550ng/sample).
However the results is very weird, because the control group gave a big differences.
I attached the result. Sample 1-9 is the control group.
Can anyone suggest why my control group is like that? The time difference during RNA isolation from sample 1-4 to 5-10 are 30 minutes.
Is 30 minutes gave us such a big differences? thank youuuu
2ul of sample is not sufficiently detailed, are you sure every sample has the same concentration? if not, you'll understand easily the difference.
fred
As Frederic mentioned, the concentration of your RNA is something you have to pay attention to. You should calculate the amount you need to add RNA based on its concentration and it cannot be all the same.
Rizki Amelia As far as you have followed the protocol precisely and used the equal amount of RNA (of same quality), then 30 mins time difference in isolation doesn't matter much. It would be helpful if you mention what is control ? and what is the purpose of this study?
Please clarify, whether control means a expression of reference genet be used for normalisation? or is it expression of target gene in untreated samples?
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