Hi,

The plat set up shown by you seems fine. However you need to do the following before starting actually on the  real-time PCR instrument

1) optimize the conditions on a regular PCR for all the genes. Design primers in such a way that you are able to get clean amplicons for all the genes at same Tm (or same PCR program)

2) See the specificity of each primer set....

3) Do efficiency analysis of each primer set..

4) I'll suggest to include one more reference gene in addition to Actin alone

Then it would be possible to the PCR setup in a way you are planning

best regards

Hi,

You have two ways to proceed here, It all depends on the number of samples you have at this point. Check if your ABI system has a 384 plate adapter that can make your life much easier. In that case you can do a plate layout establishing your standard curve for each primer set and replicates in a single plate (considering all your samples fit ).

The harder way would be do multiple plates with common samples repeated in each 96 well plate to account for variability.

Hi,

The plat set up shown by you seems fine. However you need to do the following before starting actually on the  real-time PCR instrument

1) optimize the conditions on a regular PCR for all the genes. Design primers in such a way that you are able to get clean amplicons for all the genes at same Tm (or same PCR program)

2) See the specificity of each primer set....

3) Do efficiency analysis of each primer set..

4) I'll suggest to include one more reference gene in addition to Actin alone

Then it would be possible to the PCR setup in a way you are planning

best regards

Hello Nazmiara,

Qualitative and quantification analysis of gene expression can be achieved either by using a genomic approach or a proteomics one.

As I can see here -you propose to study the expression (up-regulation/down-regulation) of 3 genes using SYBR green chemistry on an ABI platform.

My experience so far tells me that trying multiplexed reaction using SYBR is difficult and to my present knowledge ABI platforms which use either SDS software or the stepone software don't allow stratified analysis of multiple signals arising from simultaneous amplification of multiple targets - let alone a single target and a single copy gene (SCG). This however is possible if you have an access to Rotor-Gene Q system from Qiagen. Lets not talk about it much as you have already given up the idea of multiplexing (but multiplexing saves times and mastermix $$$).

Coming back to the point. If you want to quantify expression of target genes by comparing with a reference gene/ SCG; you will need 

1. Primers specific to your target - I believe 3 sets in your case

2. Primers specific to your reference gene/ SCG -  Well - dnt just stick to actin here. I agree with Ajay where he points out " I'll suggest to include one more reference gene in addition to Actin alone".  If you are wondering why he said so, its because

2.A. The efficiency with which your target gene is amplified may vary significantly from the efficiency with which the SCG is amplified under a given PCR cycling condition. So in SYBR green estimation wherein we compare Ct values to predict expression, differences in efficiency of amplification of target and SCG could decide between a  clear and a  clouded inference.

2.B.  Never pick a SCG just like that. A gene used as reference/SCG by Scientist A in his/her study may not apply to you as that gene may be effected by the disease itself in your case. So I believe its wise to dig deep and study so that you are sure that your SCG is not effected by the condition for which you are studying your 3 target genes.

I guess I could convince you here - why multiple SCG when standardizing quantitative studies.

3. I agree that a 384 plate is a relief, but the 96 works just fine (so dnt worry if you have a 96 base).

4. First aim should be to quantify primer efficiencies : for your target genes and SCGs.

Make serial dilution of your sample, add primers, master mix and run the experiment (standard curve option on ABI platform). Its always wise to have a minimum of three replicates for each sample at this point - dnt worry its not wastage of mastermix and plate but a smart investment. Your R2 values must be above 0.95 (try to have 0.98/0.99 -max is 1). Standardize this for all your three target gene primer sets. Now begin with your reference/ SCGs. Amplify using same PCR conditions as your target gene (3 genes and I am guessing three different cycling conditions) and then check the R2 values. Now since you tried with multiple SCGs, select the one which has the same/closest R2 value as your target gene (This in simple term means that under your given cycling conditions -both will amplify with same efficiency). This is the pair for rest of the study. In the end you will have three of these pairs (target gene and SCG).

5. Now you can use the Comparative Ct ( Δ ΔCt) approach and simultaneously amplify your target gene in a distinct tube and your SCG in a distinct tube - triplicates / duplicates (at least) please.

6. ABI suit compares your target and reference Ct for the same sample  after analysis and finally you will have your result.

I guess this will help you, but you have to do most of the digging - so my best wishes. Be careful with your pipetting as its really crucial in quantitative research. ABI recommends POWER SYBR green master mix  (I didn't 8) ).

And dnt worry of the plate set up. But you should know where is what. Identifying and applying unique labels can also be done post experimentation .

Thank you very much all of you. I'm getting an idea now. I have one more question:

1. Is it necessary to do real time studies for complemented sample also? I have Wild type, Mutant and complemented samples. 

Thanking you,

Nazmiara Sabnam

The Applied Biosystems Chemistry guide does a good job of explaining the system.  https://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_041440.pdf  Other replies include good cautions about efficiency, and specificity, and the difficulties of finding a good houskeeping control for YOUR system. I would like two things 1) Always include the dissociation curve when doing SYBR because ANY dsDNA binds SYBR, including primer-dimers; 2)  Verify your target identity when you are doing your initial validation.  (Melts LIKE the proper target is not quite the same as proving it IS the right target.)  Finally I'd like to caution against multiplexing.  It is a great time and reagent saver but optimization and validations (including primer limiting titrations) can be more expensive and time consuming than it's worth.  Remember that early amplfication of target A can deplete the reagents and affect subsequent efficiency of target B.  Further, multiplexing with SYBR leaves you with the problem of determining how much of the signal belongs to each target.  The old answer was "you can't tell".  Having said that, SYBR multiplexing can be GREAT for yes/no questions: Ex: is the virus present? The melting curve then shows which strain or which virus.   

You also need to have a standard curve for each gene by which you can get the gene copy number or the mass. Using the slope and intercept of the respective gene standard curve you can convert the cq values into mass or copy number, further this should be normalized with on or 2 reference genes.

Debashish has given a very detailed answer. I would go a little further and say that if you put the querry of "Real Time PCR for multiple targets"in PubMed,  you would get a  wide variety of research article to select.

Thank you very much sir.