I have run RT-PCR plate to test two primers on two diffrent cDNA (both at conc. of 500ng/microlitre) samples taken at diffrent times. The same concentration of primers (2.5micromolar) resulted diffrent amplification curve. The first cDNA resulted in a very early amplification (at around 2 treshold cycle (Tc)) unlike the second cDNA which resulted in perfect curve (after 16-20 Tc). How can I improve the reaction using the first cDNA? Should I use low concentration (like 100, 50, 25 ng/microlitre) or any other way like new calibration of baseline (if so how?),...?
Here is the pic with mixed curves
Waiting for any help...
that is too much DNA..
Usually in my hands..I measure RNA conc with RNA quantifluor kit (Promega)..I make cDNA from 1microgram total RNA (superscript III from Invitrogen) with oligodT primer (not random hexamer)..Did you calculate the RNA conc based on the Standard curve obtained by using Standard RNA?
The cDNA is further diluted 1:100 in sterile water (or 1:50.. depending on the abundance of the gene expression of your interest) From this dilution, I take 3uL for a 20ul qPCR reaction.. Typically for SyBR green, I use 50mM For and 50nM Rev primer concentration.
To check the efficiency of amplification, you should first check the amplification efficiency.. it is very important. I have attached the qPCR guide for your reference
Good Luck
Hello Geleta,
The type of curve you are saying is directly showing a contamination in DNA.. Did you run NTC for this? If yes, then what are cT values for NTC?
RNA conc. is not a problem. people do routine Real time from this conc of RNA and cDNA (that's why you observed a perfect amplification in one sample).
My suggestion is to use fresh PCR water and get rid of DNA contam.
Hope it helps !
I disagree with Ajay that RNA conc should not matter
Especially when you are repeating the expt , and then wondering why the Ct values are different. If you do not start with the same amount of RNA to make the cDNA, you will get lower Ct values in sample with higher RNA concentration (i.e no. of cDNA copies of e.g GeneX is higher in this sample as compared to the second sample with lower RNA conc.).
By not controlling the RNA conc. you are just going to add another variable. So try to keep the conditions as constant between the repeats as possible.. It will be helpful when you perform statistical analyses..
Another thing to remember is that even if you repeat the expt., some variation in the expression of genes is inevitable in biological samples. And since you are performing a very sensitive technique to analyze the expression of gene(s) of interest, you do not want to add another variation.. viz RNA concentration... (effficiency of your cDNA synthesis reaction will also matter).
In general, it is a good practice to check your cDNA with control genes (e.g GAPDH/Actin) by performing an end-point PCR before moving on to qPCR. And as Ajay suggested.. NTC is a must.
Good Luck
Well thanks guys for all the suggestions... @at Ajay, Yes I included Negative Template Control(NTC) and the Tc value was undetermined (Not shown, Under..). Having the previous results, I prepared five serial dilutions with 1:5 factor starting from cDNA of 100ng/microlitre. The five dilutions were 1:1, 1:5, 1:25, 1:125 and 1:625. These five different conc. were used to develop standard curve for the two reference genes (S24 & UBQ10). Relatively better amplification curve was seen at 1:25 for both. Three technical replications were used for both. The curves are attached here, S24 first (slope =-3.904, y-intercept =28.901, R2 =0.162, efficiency =80.349%) and then UBQ10 (slope =-9.282, y-intercept =28.404, R2 = 0.826, Efficiency = 28.154%), in order.
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