4 replies to this topic

[genie]

Hi All,
Im a newbie to real-time pcr. My task is to optimise a real-time gene expression assay for two GOIs expressed in mouse brain astrocytes. I had quantified cDNA after RT-PCR using Qubit fluorometer (Invitrogen) with their ssDNA assay kit. Has anyone used Qubit to qnantify cDNA? my first assay was done using 1ng, 5ng and 10ng of cDNA, and was able to see amplification using all concentrations of template. However, the internal control used was beta-actin, which had a higher Ct than one GOI. does this mean i have to select another internal control for my assay? how can i figure which is the best internal control, will i have to try out many? i have used oligo dT primers for RT-PCR, so can i use 18s rRNA as internal control, since it doesnt have a poly A tail? is it necessary that the internal control should always have a lower Ct than GOIs? and also one of the GOIs have a Ct of 36, can this be used in the analysis, or will i have to ignore it and understand that it was not expressed? is there an optimum Ct range for which we can use the delta-delta Ct method?

sorry for bothering you with so many questions, but i would really appreciate if any one could please help me by giving an opinion.
thanks in advance.

[Trof]

Has anyone used Qubit to qnantify cDNA?
Not me, we quantify RNA, by spectrophotometry, but fluorimeter assay should be fine if your's stnadards are OK.

However, the internal control used was beta-actin, which had a higher Ct than one GOI. does this mean i have to select another internal control for my assay?
Not necessarily, the Cts should be similar but small differences doesn't matter. What Ct values are we talking about?

how can i figure which is the best internal control, will i have to try out many?
You have to search for the genes presumably stable in your tissue and treatment and then test the stability by running them on some samples and using software like GeNorm to calculate the best housekeepers.

i have used oligo dT primers for RT-PCR, so can i use 18s rRNA as internal control, since it doesnt have a poly A tail?
You can't.

is it necessary that the internal control should always have a lower Ct than GOIs?
If you call the reference gene an internal control, then no.

one of the GOIs have a Ct of 36, can this be used in the analysis, or will i have to ignore it and understand that it was not expressed?
Can be used, it is expressed. But the variation of replicates in cycles this high may be too big and bring bigger error to your calculations.

is there an optimum Ct range for which we can use the delta-delta Ct method?
Yes. If I recall it correctly it's 15 - 30.

[genie]

Thank you so much Veteran for answering all my questions.

"However, the internal control used was beta-actin, which had a higher Ct than one GOI. does this mean i have to select another internal control for my assay?
Not necessarily, the Cts should be similar but small differences doesn't matter. What Ct values are we talking about?"

My answer is that the Ct of reference gene (beta-actin)is 20 and that of the GOI is 16. Will this affect the delta-delta Ct calculations for estimating the folds of expression?

thanks again.

[Trof]

IMHO it doesn't matter in 16 - 20, the matematics of relative quantification is same for various GOI-Reference difference, it's just if you have extreme Cts the reaction may not actually behave the same, which is needed for the calculation to be valid.
If you want to try housekeeping gene with higher abundance, then GAPDH is usually around 16 if you have 1 ug of RNA in 20ul RT reaction, but I don't find it necesary.

[genie]

Thanx for the reply, will try again with GADPH.

bye