Hi, I would like to ask how much of variation in the Ct values of the HKG among experiment and control samples is accepted. For examples HPRT gene Ct values for the control group varies from 31 to 34.7 (median 34) whereas experiment group (genotype difference for ex.) is ranging from 30.6 to 34.8 (median 31). The analysis is carried by using deltadeltaCt method, which is widely accepted and reliable. As quite similar Ct values are expected from HKGs, I am not sure of the variance in my dataset. I would appreciate if anyone can comment on this. Thanks.
Hi, variation in the Ct reflects the amount of cDNA input, so this wouldn't be a problem - although you would want to have similar input across your reactions. The values of 31 to 34 are quite high though.
Hi Gonca
I wouldnt worry about differences between bioligical replicates i.e test and control samples for your housekeeping gene. However, the differences for technical replicates for ct values within the 2 respective groups are high; what is more, the absolute ct values for a housekeeping gene are also quite high
1. You should set up reactions in triplicate and your ct values should vary between these technical replicates by +/- 0.5 cycles not multiple cycles
2. Especially with a housekeeping gene you would expect ct values of between +10 and + 25 cycles with optimal primers and especially cDNA
Given that your Ct values are unusually high for an highly expressed ubiquitous housekeeping gene and your ct differences are large I wonder if you are using too little cDNA: Did you perform a standard curve assay before performing the expression assay proper in order to determing the correct optimal dilution of your cDNA (and also check expression efficiciency of your primers). If not I tend to take neat cDNA - if reverse transcribed from >/= 1ug RNA or if you know your gene of interest is high copy number - and perform a log 10 dilution (1:10 dilution series) from !;! to 1:10,000. Alternatively, if you have transcribed from
2 things I forgot to add:
1. If performing or having performed your stqandard curve clip the dilution range so that R(2) > 0.95, reflecting a good regression fit and tight technical replicates
2. By performing reactions in triplicate if necessary you can discard one point as outlying if two concur and 1 doesnt; If the result is significant you would repeat at least once in any case
3. If the variation your referring to is actually between samples from assay to assay i.e. inter assay variation rather than between technical replicates within an assay dont worry to much as long as the technical replicates in a given assay demonstarte Ct +/- 0.5 cycles. Some variation between assays, even for the same samples, (due to things like freeze thawing of cDNA, nominla variation in platform performance) is normal
Enough said !!
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