Low Ct indicates that your amplicon many not be very abundant in your sample.   It's not necessarily something that needs to be "solved", nor is it "bad". Have others in your lab performed qPCR from the same tissue and found that the the housekeeping gene is much more abundant? Can you rule out that this is a technical error?

It's possible that your gene is just low in your sample.  You could "increase" the Ct by using more concentrated cDNA in the reaction, but then you're just scaling the reaction. It's not going to make your gene more abundant.  

If you're concerned about the high GC content of your primer, why not just design a new one? Have you checked the efficiency of your primer by producing a standard curve (concentration of cDNA vs. Ct value) or used a program like LinRegPCR?

linregpcr.nl

Article Assumption-free analysis of quantitative real-time PCR data

Dear Samantha , thank you for these advice.

The tissue type I am using is adipose tissues. The abundance of beta actin is assumed to be high for that it is used as house keeping gene.

I am wondering if changing the primer design using lower GC content can Improve the CT value. 

It is one step rt-PCR so I am not using cDNA but total RNA. The purity is above 1.8 for 260/280 ratio but low 260/230 ratio ( below 1).

I want to know if somebody had the same problem before and know how to solve it.

Dear Aisha,

If the GC content of the beta actin is high, you better do a two step RT PCR with a RNAse H treatment in between to remove the RNA from the DNA/RNA duplex, keeping single stranded cDNA as a template for your qPCR. Since the binding between RNA and DNA is much stronger than DNA/DNA duplexes.

Martin.

Dear Martin

Thank you. I cant do 2 step rt-PCR , I need to do one step.

I can redesign the primer to lower the GC value, but does this will solve the problem or it is due to rna quality?

You could try running your RNA out on a formaldehyde RNA gel to check the quality.  If there RNA is very smear-y it can indicate degradation and poor quality.  

Are you checking the absorbance ratios of your RNA on a nanodrop? If so, what does the trace look like? If there's a large peak at 230 it indicates phenol contamination in your RNA, which can negatively affect qPCR.

Dear Aisha

The b-actin gene is very GC rich in its total, not only the primer that you use, so I'm afraid one-step RT-PCR will never work.

Try another gene like GAPDH, at the end you need to use at least three different housekeeping genes as a reference in order to rule out that the expression of your housekeeping gene is changed upon your experimental setting.

Dear Samantha , thank you so much , this is true there is a high peak for the 230 ratio. I think I had a lot of phenol in the total RNA, I will try to use another method of extraction.

Dear Martin thank you so much. I will try to use another house keeping gene.

Hope to solve  my problem soon. I will keep you informed.

BW,

Hi Aisha -- 

If you choose to continue to use phenol just try and and be more conservative when removing the aqueous phase. I always try to leave at least 10% of the RNA-containing aqueous phase behind.  For example, if you have 200 ul of the upper phase, only remove 150-180 ul of it, then proceed to ethanol precipitation.

You can also use a RNA-clean up kit like RNeasy (Qiagen) after RNA extraction in order to reduce the amount of contaminants (like phenol).  Good luck!

Thank you Dear Samantha. I will try to do RNA clean up with silica membrane set. Hope to get RNA with high 260/230 value.

Best W

Hello Every body

I solved my problem. I found that my cycling conditions are suboptimal I fix it by increasing temperature and time in some steps. I got an excellent result as following :

Color Replicate Name GOI CT Norm. CT Relative Conc. Calibrator

1 26.63 22.60 1.00 Yes

2 24.59 24.41 14.32

3 22.09 18.01 0.96

The new cycling conditions are :

reverse

transcription 3) 50°C 30 min 1x

initial

denaturation 4) 95°C 10 min 1x

Denaturation 93°C 20 sec 35-45x

annealing 5) TM -5 =55-65°C 30 sec 35-45x

elongation 6) 70°C 45 sec 35-45x

Thank you for helping me.