Hi Amber,

If you are using 125 ng cDNA in a 20 uL reaction, you are using 6.25 ng/uL cDNA in the final reaction.  And, if you use 125 ng cDNA in a 10 uL reaction, you are using 12.5 ng/uL cDNA in the final reaction. 

In either case - this is too much cDNA - and you are inhibiting your PCR.

At the very most, in most situations I've seen since1998, 2 ng cDNA/uL in the PCR is the max you should use.  E.g., no more than 40 ng total cDNA in a 20 uL rxn, and no more than 20 ng total cDNA in a 10 uL rxn. You are merely adding too much cDNA template per reaction.

I have seen 4 ng cDNA/uL used with some success in one situation - but, certainly never above that in the vast majority of all situations.

Try a dilution series of your cDNA and you will see what I mean.

You have fallen into a usual caveat wherein one thinks that adding more sample per unit volume reaction will yield more signal in the end.

Just try a dilution series of your cDNA and prove to yourself that this is the case.

It seems counter-intuitive at first, but, if you realize that Taq can be "smothered" by too much template (as RT reactions can be as well), then it all begins to makes sense.

In addition, many reference and/or housekeeping genes will require even more extensive dilution before they report suitably in your PCR.  Less is more.

The concept of "reaction inhibition" is always a thing to be concerned with.  Sometimes the "inhibitor' here is the template nucleic acid itself... while in other cases it is a myriad of culpable co-extactants and/or left over reactants from prior steps used to manipulate your sample into its pre-assay status.

Lack of ample sample dilution prior to it being deployed into the PCR is the most common problem encountered across the board.  Identifying the key dynamic dilution range within which you can expect no inhibition from your particular template preparation is an absolute requirement preceding PCR.

  Therefore:  running a dilution series of your template is a good thing to pursue, up front, in every PCR endeavor.

If your target templates are too rare, then, increasing your reaction sizes to include more appropriately-diluted template per unit reaction volume as well as increasing the number of technical replicates will help you stave off the Poisson noise naturally expected from those kind of targets.

Hi Amber,

If you are using 125 ng cDNA in a 20 uL reaction, you are using 6.25 ng/uL cDNA in the final reaction.  And, if you use 125 ng cDNA in a 10 uL reaction, you are using 12.5 ng/uL cDNA in the final reaction. 

In either case - this is too much cDNA - and you are inhibiting your PCR.

At the very most, in most situations I've seen since1998, 2 ng cDNA/uL in the PCR is the max you should use.  E.g., no more than 40 ng total cDNA in a 20 uL rxn, and no more than 20 ng total cDNA in a 10 uL rxn. You are merely adding too much cDNA template per reaction.

I have seen 4 ng cDNA/uL used with some success in one situation - but, certainly never above that in the vast majority of all situations.

Try a dilution series of your cDNA and you will see what I mean.

You have fallen into a usual caveat wherein one thinks that adding more sample per unit volume reaction will yield more signal in the end.

Just try a dilution series of your cDNA and prove to yourself that this is the case.

It seems counter-intuitive at first, but, if you realize that Taq can be "smothered" by too much template (as RT reactions can be as well), then it all begins to makes sense.

In addition, many reference and/or housekeeping genes will require even more extensive dilution before they report suitably in your PCR.  Less is more.

The concept of "reaction inhibition" is always a thing to be concerned with.  Sometimes the "inhibitor' here is the template nucleic acid itself... while in other cases it is a myriad of culpable co-extactants and/or left over reactants from prior steps used to manipulate your sample into its pre-assay status.

Lack of ample sample dilution prior to it being deployed into the PCR is the most common problem encountered across the board.  Identifying the key dynamic dilution range within which you can expect no inhibition from your particular template preparation is an absolute requirement preceding PCR.

  Therefore:  running a dilution series of your template is a good thing to pursue, up front, in every PCR endeavor.

If your target templates are too rare, then, increasing your reaction sizes to include more appropriately-diluted template per unit reaction volume as well as increasing the number of technical replicates will help you stave off the Poisson noise naturally expected from those kind of targets.

Thank you very much sir for your nice suggestions