Hi,

how many cycles do you use? Have you checked that the template + reaction components is ok by running a reaction for other gene with already tested primers (you have to use reference gene anyway)? Do you prepare the mastermix or is it some comercial stuff? How do you amplify your RNA - with random hexamers? There can be many issues with a (q)PCR :)

Generally, one should also vary primer concentration, as guide, try 0.1 - 0.5 uM (final concentration). You can use much less reaction volume to save the reagents, I run 10 ul volume. But mind your pipetting technique :)

Good Luck!

Vojta

There are a few options you can try. How good is your taq? you can find a better taq to overcome the problem (I have very good experience with Phusion). Another option is maybe add DMSO to your reaction. If you have other set of primers to your template, is good idea to check, just to make sure that the template isn't the problem.

Do you know that your cells are reacting to the estradiol? If you do not have good stimulation, then the expression of TFF1 will be too low to detect and you will amplify only primer dimer.

Assuming you are doing a RT-qPCR and working on cDNA for gene expression, you may do the following:

1) check on gel or on Bioanalyzer whether the RNA you used to perform the retrotranscription was of good quality or it is degraded.

2) check on spectofotometer (Nanodrop etc) the 260/280 and 260/230 ratio for bad RNA quality or contaminants.

If the quality of yor RNA is good

3) Perform a PCR of a known housekeeping gene (i.e. GAPDH) with published primers to see if your RT works.

If all these quality control steps are ok, then probably the expression of the genes that you are studying is very low. If you think that they are sufficiently expressed, try to redesign your primers. You could also use a tissue/cell line in which the genes of interest are highly expressede as positive control. You can find this information at UCSC Genome browser (GeneSorter) or at GeneCards.

The first point is the initial polymerase activation step. You should check if 3 min of activation is enought for the polymerase you use.

Then I would think about extension of PCR product elongation step up to 30 sec/72 deg. Final extension should be better at 72 deg for 2 min. For this short poroduct ~ it should be enought. The concentration of primers you use seems to be fine.

Good luck! I believe it should work now!

There seems to be a few problems

1) What is the Tm of your primers? How did you calculate this?

2) What Taq are you using? That will help us with your cycling conditions which seem odd

All the points are valid ones. As Andrew said, the conditions are a bit strange for a qPCR of 236 bp. Normally you should only require 5-10 sec annealing for most qPCR machines and extension times of 10-15 sec (25-50 bp /sec ). The best way of getting the correct annealing temp is to make a complementary primer pair set, anneal, and do a melting curve analysis with sybr green in the PCR mix that you are using. Anneal at 5C below primer peak Tm for the subsequent qPCR. Primer Tms will vary with the mix used, so do not rely upon what is written on the tube. Antibody hot starts only need 3-5 min, but chemical /amptamer mediated hot-starts can require up to 15 mins for activation. Not knowing the abundance of the target , RT conditions, the positive control or the number of cycles, it is a bit difficult to troubleshoot further.

On a separate point, perhaps the person who downvoted the answer from Daria could let us all know what the objection to his answer is. If the RG system is to work it relies upon people giving answers freely in good faith and for the more inexperienced to be encouraged if their answers fall a bit short. Anonymous criticisms without explanation are usually not helpful to anyone and open to abuse.

Estradiol is not water soluble. Use a cyclodextran to encapsulate it. Better yet, purchase a water soluble salt.

Look adjust the Cofactor (MgCl2)

Hi all, thank you very much for your contribution and I am sorry for my late response here....

@ Andrew: I have ordered the primer based on previously published journal. and the Tm for forward and reverse primer is 55.8 and 56.8 degree respectively. I used 2x MyTaq Red mix from Bioline. I used GAPDH and it worked. The total cycle was 35. I have checked the purity of my RNA with Nano and it was good but did not run Gel for the RNA.

@ Paraskevas: I am using MCF7 breast cancer cell line and it expresses the gene. it is very well established cell line for my study.

@ Daria : can u explain how DMSO help? and how to add it in the reaction? is it instead of water? the total reaction volume is 50ul.

@ Ian Teo: Thank you for ur detail discussion. I am not really familiar with the method u described to get the best annealing temp. In our lab and also most of the lab here use gradient temp to find the best annealing temp. Can you give me any document or anything that described the method in more detail so I can try. Thank you again.

This is a pdf copy of a paper describing PCR optimisation I wrote 12yrs ago. The technology has moved on but a lot of the issues are still the same. It was specific for the glass capillary Roche Lightcycler at the time.

http://www.sciencedirect.com/science/article/pii/S0022175902002181

LightCycler qPCR optimisation for low copy number target DNA.

Teo IA, Choi JW, Morlese J, Taylor G, Shaunak S.

J Immunol Methods. 2002 Dec 1;270(1):119-33.

PMID: 12379344 [PubMed - indexed for MEDLINE]

Ian

I have some more suggestions.

1) Which cycler are you using. I use the ABI 7500 and 7900 and can give advice on optimising on these instruments

2) I know the MyTaq data sheet says it can do RTQPCR, and I do use this Taq intermittently, but I am not sure I would use it for this purpose. I would suggest the Taq that is recommended by your machines manufacturer. 

3) The other thing is that the primer pair you have in the paper may be wrong. It has happened to me twice previously that the published sequence is incorrect and I wasted time trying to get it working. Have you given thought to designing it yourself?

WIth regards to the DMSO you simply add it to the reaction mix in addition to water keeping the reaction volume the same. There are plenty of papers on the internet to help you with this, the other one which is good is betaine. Make sure you order the molecular biology grade of these reagents, and some kits that have "GC enhancers" or "Q-solutions" are simply these chemicals. 

Try DMSO and BSA, I had good results using that compounds in my PCR 

I thank you very much Teo for sharing the paper. Also really appreciate Andrew's suggestions which sounds very useful. I am sorry for my delay response to you.

# Andrew: i am using Eppendorf Master cycler Gradient for my experiment.

Hello again. 

I suppose the next step is for you to put the primer sequences up on here so we can look at them - have you tried BLAST'ing them to see if they actually align to your region of interest?

If you are using 200ng of genomic DNA please adjust to 10-1000picograms per reaction and let us know how it turns out.

@ Andrewe: yes i have tried with BLAST and i found some non specific products. but when i use another primer for another gene of interest which gave specific 1 BLAST product but in PCR i got the same result!! i mean same problem.

Hi Michael: thanks for your suggestion. did u mean 10-1000 pico gram cDNA?

@ Andrew: How can i know the recommended Taq by the machine's manufacturer? 

If you are still struggling with this I would suggest designing a new primer set. I never use published primers, I don't trust them. I always put my target into primer 3, mark an exon intron boundary to get primers in different exons, preferably flanking the longest intron. I also use a hot start taq and never get primer dimer unless it's a water only or low target level sample  in my standard curve. Designing all my primers this way means I use the same PCR program with all runs, without needing to optimise annealing temperature or Mg concentration or any other time- consuming step. About 1/10 primers give no product or more than one band or poor melt curve - I throw away and order the next primer on primer3 list.

I agree with Amanda.  I use IDT software and NIH BLAST to pre qualify primers.  In support of Amanda, I found ATCC recommended primers tO fail a BLAST test.

Hi guys, I have trued different PCR methods to get the band. still not successful. I also contacted the author of which paper I am referring for the primer i am using. He was able to get band easily. from that I guess the primer is ok. Is there any possibility to degrade the primer? I stored them in -20 degree after dissolving in ultra pure water. Is there any way to check the quality of primer?? Or any other suggestion from you?

Thank you all

Hi all,

Please advise me with this PCR method I am trying. last time i could not get any Band. Then I changed the Primer and the MasterMix. Now I got the following bands attached here. I am optimizing the annealing temperature. The band supposed to come between 200-300 bp. why i get bands below 100 bp? what is this product and how can i improve?

PCR reaction setup of TFF1/ Ps2 gene (274 bp) for Estrogen E2 (10-8 M)

Template cDNA -500 ng -0.32 ul

Primer (F) -0.2 uM -0.5 ul

Primer (R) -0.2 uM -0.5 ul

Top Taq (Qiazen) Master mix -25 ul

dH2O - -23.68 ul

Total vol. 50 ul

Reaction condition for 30 cycles

Temperature (0C) Time

Initial Denature 94 4 min

Denaturation 94 45 sec

Annealing 50 ±9 45 sec

Extension 72 45 sec

Final extension 72 7 min