Hi Dan, I don't think it's a problem of the Amplitaq gold. It's a weak amplification, that's clear. And the causes can be several:

1. Quality and quantity of your RNA (have you checked how it looks on agarose gel? If you load 0.5 ug from your UV quantification, can you clearly see the two ribosomal RNA bands at 1.9 and 4.7 kDa, the second 2.5 times more intense than the first? If you don't see them the intact RNA is too little, so it's highly degraded... If you see them, but intensity of the bands is equal, it is partially degraded, etc.)

2. cDNA synthesis (if RNA is not chemically clean, although intact, you might hinder the reverse transcriptase, which is quite a delicate, sensitive enzyme, although more and more engineered. Be sure you have no residuals of guanidine isothiocyanate or phenol in your RNA - Nanodrop UV spectra of your RNA will help you spot these types of contamination, you can check on the Nanodrop manual that you easily find on-line)

3. cDNA concentration (normally it is considered that cDNA:RNA ratio is just 1:1, so with optimal cDNA synthesis efficiency you can consider cDNA concentration to be equal to your RNA concentration in the RT. If this is true, you should obtain signal for GAPDH mRNA, which is highly abundant, with as low as 25-50 ng of cDNA in your PCR reaction - 1 ul out of 20 of the RT reaction with 0.5-1 ug of RNA)

4. Primer design and concentration (did you design primers, are they from a publication, are they commercial? Primers can be more or less or no efficient in amplifying they target template depending on their design. Your concentration could be fine with good primers, but primers that don't bind efficiently can be improved in their efficiency by raising the concentration, you can go up to 0.5 uM with a cDNA)

5. Annealing settings in the PCR cycling reaction (are you annealing at the right temperature for the primers you use? If annealing is too high, your primers will not bind or bind inefficiently. Your gel seems quite clean, you can afford lowering the annealing temperature, down to 55 °C you are quite safe. Sometimes annealing is just slow, kinetically it needs time. How long is your annealing time? You can anneal for up to 60-75 s, it will make your reaction longer, but it might improve the efficiency)

But don't be too negative, the fact that you already obtain a clean band, although weak and in some samples only, is a promising starting point. You only need a bit of optimization!

Good luck!

Hi there,

I would go for decreasing annealing temperature to 50°C and extension temperature to 68°C. Why do you perform extension at 72°C as Amplitaq gold works at 68°C (at least the Applied Biosystems one  from LifeTechnologies)? And if you are actually using the fast amplitaq you should considerably diminish incubation times... 3sec denaturation, 3 sec annealing and 5 sec extension... Here the leaflet:

http://tools.lifetechnologies.com/content/sfs/brochures/cms_052651.pdf

@Hi Pietro, thank you so much for your advice. I did carry out quality check of total mRNA on agarose gel, and all of my samples showed 2 bands of ribosomal RNA, which were very clear with the ratio approximately 2:1). The OD values of these RNA samples were quite OK (ranging from 1.6 - 1.8), so they might not degrade.

However, the random primers that I used for reverse transcription was repeatedly frozen and thawed, so I'm not sure if there is the possibility that the primers degraded and cDNA synthesis wasn't efficient. Have you ever experienced similar problems?

I started with 1ug of RNA, and after the reaction, I diluted my cDNA samples with the ratio 1:10 for GAPDH amplification reaction. Anyway, I would check the OD of cDNA again, and also the annealing temperature.

@Hi Dominique, thank you so much, I'll try to change the thermal cycle or extension temperature and see how it works. Actually, I followed the protocol of my supervisor. At the first trial, the annealing temperature was 62oC, but we obtained no band. We then tried to decrease this to 55oC, and the results were the same. I also think that something might be wrong with the thermal cycle.

Again, thank you you two so much!

Hi Dan, good that you checked your RNA. The 260/280 nm ratio of 1.6-1.8 is a bit low for RNA (would be perfect for DNA), which when clean has a ratio between 1.9 and 2.1, in water. You probably have some phenol leftover. You will easily spot it just by smelling your tube. You can clean it with one or two extractions with chloroform only.

Anyway, I think that if you just don't dilute your cDNA and use 1 ul of the RT directly you will improve a lot... 10 ng of cDNA are a bit limiting for end-point PCR unless you have a particularly efficient reaction... Random primers do degrade by thawing and freezing, as well as primers, mostly when highly diluted (working concentrations), that's why you should keep them concentrated and prepare small aliquots of working concentration primers that you finish up in 5-10 uses. But random primers are normally in such excess that this should be the least problem (how much do you use in the RT, 1-200 ng?)

Finally your fast Amplitaq... sorry, I overlooked it, Dominique is right, given the high salt/high magnesium mix they provide you, short annealing time might help and extending at 68 °C might be more efficient and less error prone (although if you don't have to clone, the latter might be less important). Be aware that for fast mixes and short cycling times you also need a very fast ramping and accurate cycler, since everything occurs faster, also unspecific amplification!

i can add very little to the excellent  and detailed answers above but the size standard in you gel starts a long way from the wells...the bands (which do look good) would look stronger if the gel were run for a shorter time and possibly a faint band might become visible as thermal diffusion weakens the visibility of bands as they run further down the gel

Hi everyone,

Thank you so much for all of your suggestions. I changed the thermal cycle and also numbers of cycle (I tried 30 cycles), and it worked. I also found out that it's not much different between diluted and non-diluted samples (according to the electrophoresis result I attached below). I used about 1ug of RNA for the reverse transcription reaction.

However, I don't know if the intensity of these bands are good enough for further analysis, since I need to detect other genes that are not as abundant as GAPDH.