In my experience, PCR can not be considered a semi-quantitative method after 24-26 cycles, even less. Doing more cycles will increase number of amplicons, therefore band intensity, but PCR efficiency is extremely variable, depending by numerous factors, among which TAQ efficiency (remember that this enzyme is subjected to temperature variations numerous times) and availability of dNTPs.

As Laura Hanson stated, you would easily detect what we are saying if you would use Real Time.

I do not suggest you to increase template amount, for the same reasons of Laura.

Firstly, I would try increasing PCR efficiency. How much MgCl2 are you using? Bring it to 3mM. Then, use 10% DMSO. Try a first PCR for 30 cycles. If you see a band, go for 25 cycles. Try 3.5mM and 4mM of MgCl2, decrease annealing of 3-4°C. If you reach a good amplification condition, it would be best. If you don't reach it, use your amplification conditions and perform a first round of 20-25 cycles of PCR, than get 1/10 or 1/20 of the product and amplify again for 10-15 cycles. In your specifical case, I would do 20 cycles of first round and 15 cycles of second round. If you continue obtaining same band intensities, do 20 cycles of first round and 10 cycles of second round.

Good luck!

Hi .. it sounds logical to me since is a semi-quantitative PCR... Looks that you have to optimize the number of cycles again. I used to do the same PCR reaction in a big volume and take aliquots at different cycles starting from 25.

In order to get amplification at lower cycles, you can try to increase the amount of your template as well..

Hope it helps. Good luck. Cheers

Hi Aditya,

It might be that 30 cycles is not enough as the expression of your gene is quite low?. I would probably do two things.

1) Run another PCR at 40 cycles and see if you get more expression than at 35, just to check that everything is in order and responding in sensible way. Also keep on eye in your controls, to see if it gives you the same result with 35 vs 40.

2) I would make and attempt to sequence the product to be sure that is what you were expecting and only one product.

Good luck!

@ francisco I did the same thing ....

@ maurcio ya i can see the increase in band intensity on the gel is proportional except for TLR4 ..I get big increase in TLR 4 band on the gel...Hepatocytes are known to express very low TLR4 as per literature..But I am getting TLR4 expression equivalent to TLR4 expression in raw cells after 35 cycles .....

Hence I am confuse which data to belive ..

Good on you!!,

So far so good I would say. Those discrepancies may be due of the low expression, hence, your controls and samples are in different stages of the amplification curve. If you have no chances of running a qPCR were you can clearly see your different amplification curves, I would go with either the results of 35 or 40 cycles. Experimentally, I do not see anything wrong, so both are legal!. However, keep in mind that I have not seen your data, and therefore is easy for me to say "either". In theory 40 cycles should be more accurate as it gives more time to the low expression samples to reach the controls or high expression samples (hopefully being both at a similar stage in the amplification curve).

Good luck!

Increase sample concentration

increase sample concentration to 1ug and change your protocol of cDNA synthesis

I agree with the suggestions posted by Mauricio, but let me voice some concerns: if a PCR reaction takes 40 cycles, then your PCR primers are not optimal. Use a different primer pair combination. If the expression levels are low, then consider a nested PCR after the initial PCR reaction. I would not trust anything with 40 cycles unless you confirm the sequence, which is a lot of TA cloning work. In summary my recommendation, trust your 35 cycle data but validate it with either nested PCR or by optimzing your PCR reaction with other primer pairs. Best of luck.

Semi-quantitative PCR are good tools to obtain a first look of your expression, But if you have a real-time PCR equipment I suggest you try a qPCR with SYBR green for instance.

This will clarify and allow you to calculate a ratio between you control and the sample.

Also will allow you to verify how efficient is your reaction, since you can monitor the precise moment in which, your sample reach the exponential and logarithmic stage of a PCR.

In another issue, if you run an end-point PCR and you control have similar band intensity at the end of 35 cycle botth (sample and control) reach the plateau in that cycle ( or before). If during the 30th cycle your sample didn't give you a band probably it was a exponential and logarithmic phase of just 5 cycle. In my experience both stages could last even 2 cycle. Thus establish a standard end cycle could be difficult and preciseness.

Good look with your experiments.

When doing semiquantitative PCR it is very important to remember that many things can affect the intensity of your final bands. Tthe relative intensities of the bands may have very little to do with the amounts of template. Once something becomes limiting in the reaciton the assay is no longer quantitative. Ifyou look at real time PCR results you will often see that replicate samples plateau at different levels, even though they crossed the threshold at the same time, or the plateau may be the same even though the amounts you started with were wildly different. You need to do a number of reactions with both you normalization control and your TLR4 primers varying the amount of template and number of cycles to make sure that, for the sample you are testing, you are in the linear/ quantifiable range. As you do serial 10 fold dilutions of your cDNA, bands should reach the same intensity 3.3 cycles appart if you have a truely quantitative system. As several people suggested, if you want to use the same cycle number for both your RAWs and your hepatocytes you will need to use more template from your hepatocytes. Of course using more template can be risky as you also can increase the amounts if inhibitors present in your sample- so it is extremely important to do the serial dilutions of both your cDNA from your RAW cells and your cDNA from your hepatocytes to determine the conditions (if any) which allow you to get quantitative results.

In my experience, PCR can not be considered a semi-quantitative method after 24-26 cycles, even less. Doing more cycles will increase number of amplicons, therefore band intensity, but PCR efficiency is extremely variable, depending by numerous factors, among which TAQ efficiency (remember that this enzyme is subjected to temperature variations numerous times) and availability of dNTPs.

As Laura Hanson stated, you would easily detect what we are saying if you would use Real Time.

I do not suggest you to increase template amount, for the same reasons of Laura.

Firstly, I would try increasing PCR efficiency. How much MgCl2 are you using? Bring it to 3mM. Then, use 10% DMSO. Try a first PCR for 30 cycles. If you see a band, go for 25 cycles. Try 3.5mM and 4mM of MgCl2, decrease annealing of 3-4°C. If you reach a good amplification condition, it would be best. If you don't reach it, use your amplification conditions and perform a first round of 20-25 cycles of PCR, than get 1/10 or 1/20 of the product and amplify again for 10-15 cycles. In your specifical case, I would do 20 cycles of first round and 15 cycles of second round. If you continue obtaining same band intensities, do 20 cycles of first round and 10 cycles of second round.

Good luck!

The MgCl2 conc I am using is 2.5mM , I also tried with higher conc but not much difference in the results..Then a used the Promega supermix also which gave me better result compare to individual components.. I did tried 25 cycles 30 cycles and 35 cycles...25 cycle the expression level is too low and with 30 cycle good expression for my positive control and my other TLR's but with 35 i can see suddenly TLR4 band shoots up..

But I can try with realtime PCR as mention by others...

.

If you already use 2.5mM MgCl2, I would also try DMSO 10%. Believe me, it really increases PCR efficiency. In my experience, it is nor true that it helps just high GC rich amplicons. I use it in every PCR mix. You can easily get an aliquot from cell culture labs, which use it for cell freezing. Get 1ml of DMSO 100% and put in a 1.5ml eppendorf. Use aluminium foil to cover the eppendorf since DMSO is photosensible. Store it at room temperature in a drawer. Every time you use it, vortex it for 10 seconds before adding it to your master mix.

please explain the logic of selecting number of PCR cycles for semi-quantitative PCR for any gene in question..??

Sometimes the number of cycle post 35 cycles may amplify non-specific product. Hence it is always advisable to have cycles less than 30 if possible or 35.

Thank you sir...