• It is possible to assign a particualr sample group the role of calibrator (expression of '1') and therefore compare expression of other treated samples to this group using delta delta ct analysis
• However, I think Vincent makes a very good point: If your Ct values are greater than 30 then that implies low expression and you would need to be absoluultely certain that triplicates are concordant (+/- 0.5 cycles) before trusting data in this range
• Regarding samples with ct values of 40 that falls into a region that is effectively noise and so it makes no sense what so ever to trust such values
• When I have dealt with patient samples associated with limited amounts of material and/or very low expression (as your Ct values indicate) I have added a pre amplification step after RT before performing qPCR: If you would like me to send you a protocol I can

Hi,

You should first take a look to your housekeeping gene CT .

A CT above 30 make no sense, it should be in the range of 22-28

Then, the CT in your patient groupe above 40 may indicate that you have almost no CDNA in your sample.

Can you give more information about your RNA extraction in patient and healthy ?

thanks Vincent for your comments. it is my first trial in this work. i understand your comments. but i want to know what about delta c of calibrator if you can help me

• It is possible to assign a particualr sample group the role of calibrator (expression of '1') and therefore compare expression of other treated samples to this group using delta delta ct analysis
• However, I think Vincent makes a very good point: If your Ct values are greater than 30 then that implies low expression and you would need to be absoluultely certain that triplicates are concordant (+/- 0.5 cycles) before trusting data in this range
• Regarding samples with ct values of 40 that falls into a region that is effectively noise and so it makes no sense what so ever to trust such values
• When I have dealt with patient samples associated with limited amounts of material and/or very low expression (as your Ct values indicate) I have added a pre amplification step after RT before performing qPCR: If you would like me to send you a protocol I can

yes please, Laurence , i will be happy and thank full to receive your protocol. and please explain for me what you said about role of calibrator.. thank you so much

Hi. Noha

Delta Delta Threshold cycle (ΔΔCq) expression values were calculated for RNA samples of each treatment to determine genes expressions using (reference gene) and the sample control.

Δ Cq = Cq – reference gene.

ΔΔ Cq = Cq – control.

ΔΔ Cq expression = 2 (-ΔΔ Cq) .

The equations show the mathematical model of the relative expression ration for the real time PCR. The ratio of the target gene is expressed in sample versus control in comparison to reference gene.

Good luck

Thank you dalia, my study is not treatment study but case- control study (patients and healthy persons).. i want to compare between specific gene expression between patients and healthy. so i need to calculate ΔΔ Cq expression for each patient and each healthy person. i can easily calculate Δ Cq but i need Δ Cq for calibrator to complete the equation. i hope you get my problems..thanks again dalia

I know but i want give you example, you need used a reference gene in this study to calculate Δ Cq. from this equation: Δ Cq = Cq – reference gene. Whereas, Δ Cq = Cq for each (patients) – reference gene. Also, Δ Cq = Cq for each (healthy) – reference gene

so you well get Δ Cq for calibrator to complete the equation.

Welcome

• As demonstrated in the example provided, assign the control patients an arbitrary value of 1 in terms of their fluorescent signal and therefore level of gene expression (normalised to house keeper control) - therefore they are designated calibrator - and therefore express the expression in treated patients as a fraction of this expression
• You might find a previous answer I provided useful:
• https://www.researchgate.net/post/How_can_I_calculate_gene_expression_levels_with_RT-PCR?pli=1&loginT=DfXNfcV2LWrxS29D_VXaENq2aBlYcxf_0zheWCb7zt1SoKnYAS8CBDTqL2pINOLyidamUuYeNmn3NDomswub-FormIU&uid=UyKBojZHrVWO1sIjqLaRGmsoovN0kAVtlBIO&cp=re68_x_p2&ch=reg&utm_medium=email&utm_source=researchgate&utm_campaign=re68&utm_term=re68_x&utm_content=re68_x_p2#view=54994b14cf57d76a568b45a3

thanks dalia too much, i have used a reference gene. but i hadn't calibrator.. this is my problem...some colleagues suggested to use the the sample with the highest Δ Cq to be my calibrator and to be subtracted from all the patients and healthy persons to obtain Δ Δ Cq ..i want to know is this true or not??

thanks so much Laurence for your answer. i have red the previous question and your answer but i still couldn't understand the step number 4 in which you said

'" 4. Now take these 2 cycle  or delta differences and take one delta value from the other: This is where the term delta delta is derived from "

please, could you explain it by small example in excel sheet if you have a time. thanks again for your help

• Sorry Noha
• Subtract the delta(Treated Ct value of the gene of interest -housekeeper) from the delta ct value of control (i.e. Ct untreated GOI -Ct untreated housekeeper)

Hi, according to Livak equation

ΔCt= Ct gene of interest – Normalization Factor (a)

Normalization Factor is the arithmetic mean or geometric mean of Ct values of the selected control genes. If multiple genes are selected as controls, a gene stability measure is also calculated based on the geNorm

Algorithm to assist with selecting most stable control genes for data normalization . The normalized ΔCt data are used to calculate the relative gene expression fold change using a selected calibrator (reference sample):

ΔΔCt= ΔCt sample A – ΔCt calibrator (b)

Fold Change = 2-ΔΔCt (c)

i suggest you to read this ppt and good luck

Presentation Basic of quantitative real time PCR