cellthetruth

Dear all,

I am confused about some sensitivity/gain - background issues.

We use a dual luciferase based assay system to measure gene expression. Now some of the genes are expressed very high, some are expressed very low. The latter show so low values it seems to be below background values, no matter what blank measurements I make (air, water, buffer+substrate).

From the literature I know the principle how photomultiplier tubes (PMT) should work. Therefore, a signal increase seen by increasing the gain is an artefact, i.e. increasing gain will not increase the input.

However, when I compare the measured signals with our expectations/hypothesis, meaning the regulation pattern of the genes, it is as expected. We repeated these experiments at least 10 - 20 times and it is highly significant by t-test statistics (as well as by looking at the data by eye). So the results fit our model, but in theory they shouldn't... Correct?

How can this be explained? Has anybody a good idea, knows some good literature? Are we producing simply artifacts by chance?

Thank you for any help to make me understanding,

cheers

Hi,

I want to do qPCR on a gene which is encoded on a plasmid. I have HEK cells transfected and subsequently isolated RNA and digested the extract with DNase. Before reverse transcription I perform a control pcr to check there is no more plasmid present. This is how it should be... Is this possible? How big is the risk that there is still some plasmid left and therefore invalidate my qPCR results? Any tricks or special things I have to take care of?

Thanks

Hello everybody

i'm doing a qRT-PCR for the first time in my lab-career. My supervisor told me I need to do an RNase H treatment after cDNA synthesis. But I don't understand why!??? The DNA polymerase should'nt be able to amplify my GOI from RNA, that's the reason for cDNA synthesis. Or am I wrong here? Is there any other reason?

Thanks for help / explanations

Hi there,

does anybody know how many nucleotides the longest mRNA imported into the mitochondria is? I know from 5S rRNA but this one is not translated and not very long.

couldn't find anything specific in the literature. Any ideas?

Cheers

Hi,

I have the problem that over the weekend my cells did not grow. We started to use Mouse Embryonic Fibroblasts (MEF) and observed last week they grow  nice in DMEM + FCS. Now after only two passages they stopped growing. They are not dead but won't proliferate anymore. How to explain? Is there a medium other than DMEM suitable for MEF? Usually I work with HEK and NIH3T3 cells which both like DMEM.

Thank you, cheers