Hi,

you could do a comparative qPCR, in both sybr green that  taq-man probes

I prefer taq-man probes! if you need the protocol, you could write and I will send the protocl that I normally use. You have to choose the reference gene and the reference sample. Otherwise I use a qpcr absolute quantification with a standard curve.

Let me know and good job.

Bye

Hi,

you could do a comparative qPCR, in both sybr green that  taq-man probes

I prefer taq-man probes! if you need the protocol, you could write and I will send the protocl that I normally use. You have to choose the reference gene and the reference sample. Otherwise I use a qpcr absolute quantification with a standard curve.

Let me know and good job.

Bye

Thanks so much Celestina for your answer

we hav in the lab. both syber green and taq-man probes

it will be great if you send me your en house protocol

i will use GAPDH as the house keeping gene

but the reference sample, you mean the negative control without any therapy?

i did not extract RNA from a negative control cell line, i only extracted from the comparison arms only !!

but you are right i should have a reference sample

qPCR absolute and comparative quantification volume 20 microliters.

10 µl master mix (10X)

1 µl of probe (20X)

1µl cDNA (35nanogrammi)

H2O to 20µl final volume

all the unknown samples in triplicate for each gene plus negative samples in triplicate for each gene.

For each gene you have to create the standard curve (7 or 5 points) by serial dilutions 1: 10.The instrument STEP plus Applied does not require the GAPDH because you can measure the levels of expression of your sample directly on the curve standad gene. example gene X: you construct the standard curve using the cDNA of a control on this curve and measure the expression of the gene X in your sample. Otherwise you can make a standard curve of the house keping gene, I use the 18S, and evaluate your samples (2 to 5 different genes) on the 18S.

If you want to use the comparative qPCR, I always do a comparison of the expression of the same gene in two different samples: es y gene in sample 1 vs 2 sample compared to the gene 18S as house keeping Sample 1 and Sample 2.

What  instrument  have you in your lab?

I hope to be of help ....

Merry Christmas

Sorry: my mistake.

The protocol is:

qPCR absolute and comparative quantification volume 20 microliters.

10 µl master mix (2X)

1 µl of probe (20X) if probes contain F and R primers or 1 µl of probe (20X) without primers, so add primers 0,6µl  F and 0,6µ R

1µl cDNA (35nanogrammi)

H2O to 20µl final volume

all the best

Thanks so much Celestina

i will follow your protocol

you are great teacher and scientist!!

i appreciate expending your time helping me

All respect and Best wishes

THANKS!

See you soon

Ciao

I would suggest that you could would better run a test run of qpcr to check the quality of cDNA before you start with the actual 5 genes. You could also figure out which house keeping genes gives you the least Cq value. So try out GAPDH, B2M, 18S. Whichever gives you the least Cq would be the best housekeeping gene of choice. 

Doing triplicates would give you better trend.

5 genes or targets, one more target for housekeeping gene, 2 different sample and  triplicates. 6 * 2 * 3 would mean you would require 36 wells.

I would suggest you could make a master reaction mix for each gene with a little extra to reduce loss due to pippetting error. Which means you would make your reaction mixture for each gene as following in case of triplicates

70 ul MM,

56ul dis water, if in the case of 20x probe stock, if it is 10x probe stock make it 49 ul dis water

7ul probe mix if it is 20 X probe stock if 10 X probe stock make it 14 ul.

Now the total voulme would be 133 ul mix it well and pippette out 60 ul into 2 different tubes and  then add 3 ul of different cDNA sample into the two different sample tubes or the 2 different 60 ul tubes and mix them well.

Then load in each well 20 ul for the triplicates.

This is how I do at my lab.

Thanks so much  Raghuvaran Narayanan, i appreciate this very precise and organized protocol so much

you are a great help to me

Best

No problem Anytime

One more advantage of doing a check for which housekeeping gene you would want to select i.e if you are going to run b2m, gapdh, 18s is that you get to know the quality of your CDNA how good your RT kit works. You reminded me of this by endorsing my Reverse transcription skill thanks for that...

i can not describe how your protocol helped me in my expeiment....!!

it is more than Excellent,i appreciate your sincere help....

Best regards Dr.Narayanan