In realtime PCR, I have some confusion about 45 thermo cycle or 40. What is more benefit? When do we run with 45 cycle?
You increase the cycles in a PCR reaction (not only Real-Time) to increase the double-stranded DNA fragments produced.
I personally do a slight increase on the cycles when the initial DNA product is not much (low concentration) and results in faint bands (on Gel Electrophoresis for normal PCR).
Hope this helps.
Dear Lee
- It really depends on the copy number of your gene; in other words at what level it is expressed
- Generally speaking, most persons opt for a maximum of 40 cycles because above that number if you cannot detect your gene of interst then you are approaching the limits of detection of the machine. In other words, Triplicate samples tend to be less concordant in this very low copy number region on account of additional noise
- Indeed, most people in general do not trust Ct values above 35 (hence the 40 cut off)
- Ultimately however it does depend on how efficient and effective your primers are to a particualr target region of a gene:
- In other words, if you perform a serial dilution and then plot a standard curve then providing the regression lines still results in a good fit, i.e. R(2)> 0.98 and the efficiency value based on the slope of the line is > 85% then it valid to say your reaction is still within dynamic range at or above 35 cycles (hence the 40 cut off)
- In addition, to perform accurate gene expression analysis you need to compute the mean of 3 4 technical replicates and at Ct values above 35 (and invariably and definitely 40) the spread of Ct values is > 0.5 cycles and consequently the variance is too high to accurately cimpute a valid Ct value
- Just to go back to your original question, if the expression of your gene was such that Ct values were coming in (say) at 41 cycles necessitating a cut of 45 then based on serial dilution and this curve it is very unlikely your data would be within dynamic range
- In other words for most gene primer combinations, there isnt much point setting a cut off of 45: For genes above 40 cycles Ct it is custmary to perform pre amplification of your RNA before making your cDNA to bring your data back into dynamic range as described
- Find attached a document that describes how to calculate dynamic range for your cDNA primer combinations
Dear Le
In real-time PCR, reactions are generally run for 40 cycles.
Hi, If you follow a standard protocol of any qPCR mix kit provider, you really don' t have to run to 45 cycles. I have recently worked on qPCR data analysis methods and would like to share soem ideas with you. If you run more cycles to get to the linear range to meet the requirement to use delta-delta Ct method for transcript quantification, probably due to low abundance fo your target genes, then your results will contain more accumulative error, meaning your data will turn to be more inaccuracy. To add more template in the PCR reaction mixture will enhance the appearance of the amplification fluorenscent intensity which can be recognized above the background, refered as 'outlier', so that the outlier can appear earlier, consequently, more accurate template abundance will be obtained due to less accumulative error involved. I guess to run to 40 cylcles is only to secure some data above the background in the case that your target abundance is extremely low.
I do recommend 40 cycles only, if you have theoretically just one molecule of the target DNA it will be amplified and detected easily before reaching cycle 40th.
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