Hi Rawad,

Some of this is kit and scaling dependent. If using a reverse transcription kit, what does the manufacturer recommend? I generally use the BioRad iScript reverse transcription kit in a 20uL reaction volume with 1ug of RNA. I then dilute the product 1:5 with water and use 1uL or more for qRT-PCR (volume used depends on how abundant the gene of interest is, but most of the time 1uL is enough for me).

I would be hesitant to include too much RNA in the reverse transcription step, as this step has limiting reagents that could introduce some bias in the abundance of transcripts after reverse transcription. However, as long as your Ct values do not get too low (gene is too abundant), you can load quite a bit into the qRT-PCR step (for example I have used 7uL before). Your Ct values are not unreasonable (assuming this is not the value for reference genes), so is there some reason you want to have more RNA?

Thank you Jessica

Am using maxima enzyme, cDNA kit and their recommendations is from 1 pg to 5ug, I thought if I load more of cDNA, it might lower my ct values, so I will have more gene expression we are working on complements ( c5a, c5ar)in hypoxic,normothermic rats

My house keeping gene is Gapdh with a ct value of 18 

Thank you for your help

I guess I wouldn't have any concerns with the Ct values you are getting now, but it sounds like you could at least double the amount of RNA in your reverse transcription, which would bring your Ct value down by 1. But by loading more cDNA in your qRT-PCR you can bring it down more (for example by using 8uL cDNA the Ct value will decrease by 3). An additional benefit is you would not have to use more cDNA in the reference gene and worry about having too low of a Ct value.

Thank you

Hello Rawad,

two quick points:

1) Can you please report the total volume of your RT reaction? this information is important to calculate the concentration of cDNA (in terms of nanograms/ul of starting RNA) that is present in your Q-PCR reaction. E.g. if you RT 1500ng in a total volume of 20ul, this means that, by using 1ul to perform the Q-PCR, you are actually using 75ng of 'RNA' for each reaction. Instead, if you RT in a volume of, say, 40ul, 1ul of RT will contain 37.5ng of 'RNA' and so on.

2) Unless there is a specific reason not to do so, I prefer to use more than 1ul for a typical Q-PCR reactions. Using small volumes of template almost invariably causes pipetting inaccuracies (error is always higher with small volumes, even with good quality pipettes; also the amount of template solution that is present in the outer part of the tip, which you can consider as a costant, will 'weight' more if you use small template volumes) therefore leading to lower reproducibility (higher standard deviation in your replicates).

hello Rocco

the total volume of my RT reaction is 20ul , thank you for your opinion , we started recently working with q-pcr

my other question  if i increased my cdna concentration in a specific gene does this apply for my reference gene as well 

thank you 

Hello Rawad,

I'm not sure I fully understood your question: assuming that your RT is done using classical techniques, such as random hexamers, oligo-dT or a combination of both (i.e. NOT using gene specific primers for the RT reaction), what you end-up with is a reverse transcription occurring, ideally with similar efficiency, for all the genes that are present in the RNA template (there are significant differences between random hexamers and oligo-dT: rRNA, 5' of long genes and so on, but this is another story..), therefore comprising both gene of interest (GOI) and  housekeeping. So what you expect when you increase the amount of cDNA per reaction is a proportional decrease of the Ct for both your GOI and the housekeeping. Example: using 1ul of cDNA you detect your GOI at 25 and your housekeeping at 18; by using 2ul of cDNA you expect your GOI to shift  to 24 and your housekeeping to 17 (assuming, for seek of simplicity, that Efficiency E = 2 for both target and housekeeping). Given that relative expression is basically a ratio:

E^Ct_Housekeeping / E^Ct_GOI

and assuming E = 2

2^Ct_Housekeeping / 2^Ct_GOI

So, for 1ul reaction:

2^18 / 2^25 = 2^(18-25) = 2^-7 = 1/2^7

which basically means that, assuming identical amplification efficiency, your GOI is expressed 2^7 fold less than your housekeeping.

Now, the 2ul reaction:

2^17 / 2^24 = 2^(17-24) = 2^-7 = 1/2^7

Again, exactly the same result, which makes sense because here you measure the RELATIVE expression of your gene normalized against the housekeeping.

Therefore, the typical reasons why you may want to change the amount of cDNA in your Q-PCR reaction are:

1) Ct of GOI or housekeeping are too low (e.g. less than 10-15 depending on your instrument and baseline settings) -> less template

2) Ct of GOI or housekeeping are too high (e.g. more than 30-35, again depending on your instrument) -> more template. In this case a good indication of whether you need to increase the amount of cDNA is the analysis of the standard deviation of the sample(s) with Ct > 30. As a rule of thumb, if StDev is ok you don't need to repeat.

Hi Rawad,

You do not need to use the same amount of cDNA between your gene of interest and your reference gene, but if you want your calculation to be a true relative expression calculation like Rocco described, you need to account for the dilution/concentration factor used. For example, if you use 8uL for your gene of interest and 1uL for your reference gene, you would either adjust your gene of interest Ct values by adding 3 or divide your relative expression calculations by dividing by 3.

If you are only interested in fold change between your samples, no adjustment is needed.

OK thank you

Rocco and Jessica, that was very informative  

Happy new year

The attached book chapter may have some useful ideas.

Thank you Jack

@Rocco

Hi Rocco,

I am facing a similar issue. The ct for my housekeeping gene is 25 and for GOI is 27 in the Control sample and 29 in the treated sample. I have used 2ug RNA to make cDNA and use 2.5ul of (1:1 diluted cDNA) in a 10ul RT PCR reaction. What can I do to decrease the Ct. Shoud I scale up the starting RNA conc.

Thanks

Regards

Renu Bisht

Renu Bisht

When you dissolved RNA in TE, EDTA act as a chelating agent to remove divalent cations Mg2+ which could affect your PCR. To increase the RNA amount you could use any of the following options.

1) Using RNA concentrator to concentrate the RNA and use a high capacity Reverse transcription kit.

2) If your RNA is free of DNA you could avoid DNase treatment and able to use one-step RT-qPCR kit

3) You could change your qPCR mix to which optimized to give low Ct values.