Remember for PCR, Cts are mostly influenced (for optimized reactions/mixes) by the starting concentration of the template. As such, you generally try to improve parameters retaining to that:

Could try to add more input for cDNA reaction (i.e. instead of 100 ng of RNA try 200 ng RNA) but be careful to not overload the reaction. Every doubling of input would improve Ct by 1 so that if you had used 100 ng and got Ct of 32, 200 ng of input would give Ct of 31.

I'm not sure what the recommended cDNA dilution of your kit is, but if permitted 1:20 dilution could be worth a shot (it would improve your Ct by less than 1 Ct). Going under the recommended dilution could be inhibitory to the reaction even though you will be giving more input template.

Using temperature gradient optimization could be worth a shot too. Optimized annealing temperatures can give a decent boost if you chosen Ta is suboptimal (see fig 2.11 in https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5279.pdf).

Sometimes commercial master mixes with optimized chemistry can also help as well, in my PhD thesis I tried some home-made brew PCR reaction (with our own buffers) and didn't get good results for the microRNAs that I screened. Tried the same experiment with a commercial mastermix and got much, much better results.

Would also be good to give input that is relatively free of inhibitory molecules to minimize the effects of carry of contamination which can be inhibitory (a good profile of A260/280 and especially A260/230 for chemical contaminants).

I wouldn't expect the primers to be a problem as long as you are working within the range of general recommendations for your chemistry. As long as you aren't undersupplying or oversupplying. One other thing that could help out is using triplicates, I know when you have Cts that are around 30-35, your Ct values can have high standard devs.

Nothing wrong with a Ct in the 30's but if you're expecting even lower target DNA content in other samples you'll be struggling to detect it

The primer concentration should be determined by the kit/ mastermix you're using. For example, the QuantiTect SYBR green from Qiagen recommends 0.3uM primers. You can titrate these upwards but it's probably not the primary issue.

Is there any reason you've diluted your samples so much and when you say the 1:10 didn't help much, what Ct did this decrease to? As Can said, Ct should decrease by 1 with a doubling of DNA.

Possibilities to consider:

1. Is your template input just really low. You can quantify total DNA input using the Qubit or Tapestation. You can use a Nanodrop as last resort. Given you're pooling samples, is there any reason you would expect higher Cts?

2. Have you introduce contaminates during sample prep. Ie residual ethanol is your samples or nucleases.

3. Have the samples and/ or reagents been freeze-thawed too many times.

Things to list for future help:

1. DNA/ RNA input.

2. Kit you're using.

Your qPCR is probe based or dye based?

You quantification is relative or absolute?

You want to decrease your ct values?

What you need in qPCR is the sample should be exponentially amplified and should reach plateau.

Hi,

Ct values in the range from 32 to 35 means low expression of the target genes in your sample. Ideally, Ct values should be between 15 to 30. Anything above or below this range is not reliable and means that you need to adjust the template concentration.

Initially, before multiplexing your samples, you need to spend some time running a few qPCRs just for optimizing the template concentration, primer concentration and annealing temperature.

If you are using a master mix, it will usually be 10x, and this is fixed, and you only need to tweak the other above mentioned factors. If you have followed GLP, aliquoted your master mix, stored at -20C and thawed the master mix on ice, then your Taq polymerase in the mix should be fine, and you can rule out any issue with the mix.

What about the Ct value of the endogenous/housekeeping gene?

Try amplify a known tissue-specific gene as a positive control.

Since melt curves show single peaks, you can go ahead and increase the primer concentrations in 0.2μM or 0.5μM increments in an 8-well strip, and observe for any linear decrease in Ct values across wells.

If your sample is not limiting, see that you use at least have 10ng of cDNA template in each tube, during the initial optimization.

Above all else, see that your RNA template that you have used in the reverse transcription reaction was isolated efficiently and has a 260/280 ratio of ~2, indicating pure RNA. But for accurate quantitation it is best to use the Qubit fluorometer to calculate the right amount of RNA template as input for the RT rxn. Based on the conversion capacity of the RT kit you are using, the amount of RNA template will vary.

Also, see that once you complete the optimization, you should use the same brand of RNA isolation kit and RT kit for the whole experiment.

hope this helps.

Finally figured out what's causing high Cts. I'll share it here.

It was the Extension temp. I was using a 3-step protocol (separate extension at 72C from annealing) according to the manufacturer's suggestion for primer Tm < 60C. The 18S ref gene did not have any problem with the 3-step, only the GOIs. After tweaking cDNA input, primer conc., and annealing temp without success, I decided to revert to the standard 2-step qPCR protocol (annealing/extension at 60C), and then the Cts all went down to 20's. This was a surprise though since I've read that the 3-step protocol is supposed to increase amplification efficiency, with Taq operating at its optimal temp. How come a single annealing/extension temp was better?

I am using PowerUp SYBR Green Master Mix by the way.

Thanks to all those who suggested answers.

If the manufacturer recommends that the protocol is done within certain parameters, then it ought to be done that way. If it is not working with the protocol that they gave, then it is a problem that they need to deal with. The 72*C extension is sometimes necessary for longer amplicons to get the desired yield. Two-step is OK if you are within the parameters of two-step PCR (see https://www.thermofisher.com/tr/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-cycling-considerations.html).