Real-time PCR low efficiency

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anax
anax's picture
Real-time PCR low efficiency

Hi everyone!
I am new here Hope someone can help me!

I am doing quantitative real-time PCR to determine mitochondrial DNA genomic copy number (for the ones that are not familiar with mtDNA: copy number varies from cell to cell and with different conditions). I am using SYBR green and standard curve quantification and have already done this with success in the past. However, I changed to another lab and am now having problems.

I am doing exactly the same I was doing, but with a different machine (past: Corbett; present: BioRad) and different master mix/SYBR reagent (past: Quantace; present: BioRad). The reactions look nice: high r square (99%), no contaminations, and nice melt-curves. However, efficiencies are always low (75-85%). Indeed, from one standard to the next (10-fold dilution series) the Ct always increases 4 units (which is too high). It seems that I have inhibitors in my reactions. However, the samples I use to create the standards are isolated through base-column assay. I have already tried different primers concentrations and annealing temperatures and times. At last, I increased the extension times and got 2 reactions with 95% efficiencies. But next reactions got wrong again!

Should I try different primers? If so, which primer-design program should I use? Is the one from Invitrogen available online good? Or should I use a purchase-one?

Thank you in advance. And sorry for being so long

R Bishop
R Bishop's picture
I routinely use the

I routinely use the Invitrogen Online program with great success. I have never used the biorad qpcr setup though. You might at least borrow a sample of SYBR master mix from another lab and compare the two also, try a rxn in another machine. Our qPCR machine stopped reading well in a few wells and it took us a while to realize what was going on. Hopefully others have ideas for you on the Biorad assays.

Best

Rb

anax
anax's picture
Hi! Thank you very much for

Hi! Thank you very much for your help!

beatris
beatris's picture
Hi,

Hi,

I have the same problem you described in your post.

I was wondering how did you solved it.

Best,

Beata

Biju
Biju's picture
Hi Beatris

Hi Beatris
My experience is that commercially available primers (Taqman ) will do superior job compared to SYBR chemistry with regular primers.
Alternatively you shoud try to stick to same thermal cycling programs as already established in the lab.Sometimes trying a different cell line will also give a better standard curve.
Lastly I will check the time lapsed after the purchase date  in case of Taqman primers. These primers could loose potency due to repeated opening, feeze/thaw cycles even though ABI does not give any shelf life or expiary for these primers. Covering the Taqman primers  with aluminim foil also seems to preserve the potency of the primers.
Hope this helps to some extent.
Biju

beatris
beatris's picture
Thanks,

Thanks,
 I do use the pre-desighned primers, but unfortunately it does not help. The eficiency is low. It worked well before and than just stopped working. Could it be the problem with lamp or some other intrinsic features of the PCR machine?

Biju
Biju's picture
Hi Beatris

Hi Beatris
First I do not work with mitochondrial Real-time PCR. If some thing uinque with mitochondrial expts. I cannot help.
 I will try the following.
1. I will check with others if their reactions are working on the machine in doubt. If nobdoy else is using this machine, I will set up my reaction in another real-time PCR machine with my primers OR set up a reaction with a primer from another lab (which has consistenlty worked  before) in the machine in question. This will answer whether the machine or  so ehting else is in question.
2. I will check my cDNA for quality by nanodrop.Thsi will tell whether the cDNA has degradded or not.
3. I will run the diagnostics test for the machine and also call the service technician to ensure that everyhting is fine with the machine.
Biju

pawan123
pawan123's picture
I am doing quantitative real

I am doing quantitative real-time PCR to determine Micobacterium avium paratuberculosis IS900 gene copy number I am using SYBR green and standard curve quantification The reactions look nice: no contaminations, and nice melt-curves. However, efficiency are always low (40-55%).Indeed, from one standard to the next (5-fold dilution series) the Ct always increases 4-6 units .The samples I use to create the standards are isolated from Culture and its conc. is 28ng/ul.