8/19/2023 0 Comments RtcontrolsWith dUTP, the PCR product will have U incorporated into the strand. If your contamination is a PCR product, you can try using dUTP in the qPCR mix. That should melt the dimers so that you only read specific signals. Right after annealing, you can add another data acquisition step at a higher temperature than the extension step. You may also see this in the high-dilution wells since there is little template. It is common to see dimers form when there is no actual template for the primers to work on. Q I don't dilute the RNA, but instead reverse-transcribe and then dilute the cDNA.Ī Your NTC signal might only be from the primer-dimer product, so it may actually be negative. That could cause variability if different samples have varying levels of contamination.Īfter you make a batch of cDNA, do you dilute it for qPCR or do you dilute the RNA and then perform the cDNA reactions? I recommend the first way. If you see a peak in your water control, then you have contamination. ROX is not going to do that since it doesn't bind DNA. As the temperature goes up and melts the strands, the SYBR is released and stops fluorescing. The melting peak is caused by the change in fluorescence as SYBR green binds dsDNA. I tried running the genes in different positions on the plate and still got variable results, so it doesn't seem to be a problem with the machine.Ī The ROX channel cannot have a melting peak. The instruments are monitored by the technician at the core facility and other users haven't reported problems. I tried running a few plates on an Opticon machine but got similar results. I see melting curves for the ROX channel, the NTC, and the NTC SYBR Green signal. It helps a little in correcting the signal, but the results aren't affected much by its presence. Is your instrument calibrated and running properly? I get very tight replicates using the Qiagen SYBR green mastermix. ROX dye should normalize for differences in the mastermix. Qiagen supplies this option with the QuantiTect reverse transcription enzyme and Bio-Rad offers it with their iScript enzyme.Ī Unacceptable replicates are usually caused by pipetting errors. If the problem is with the cDNA as you suspect, you could try using a mix of random primers and oligo(dT). If this is your problem, you might try reducing the amount of primer you use. How can I solve this problem?Ī The amplification you are seeing may actually be primer dimer and not real amplification. I guess the variability is coming from poor-quality cDNA. I verified that there isn't genomic DNA contamination by PCR with suitable primers. I checked every step on a gel and sequenced the PCR products. Adding 2.5% DMSO helped very little and using more DMSO inhibited the reaction. Because of this, I was very careful with the primer design and tried several primer dilutions. The genes form secondary structures and the genome is duplicated. I am using the MX3000P system from Stratagene and SYBR Green II. I reverse-transcribe using Superscript II and oligo(dT)20. I tried total RNA extraction and using Dynabeads from Invitrogen and prefer the latter because I get better-quality RNA as determined by Nanodrop and Bioanalyzer. I see amplification in the no-template control (NTC) reactions and unacceptable technical replicate differences in the Ct values. Q I am having some problems with qPCR using plant RNA. How can I avoid technical replicate variation and signals in my no-template controls? (Thread 21619)
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