I am doing expression profiling of different miRNAs in breast cancer patients. I did qpcr analysis using GAPDH as housekeeping gene and got GAPDH values that were varying considerably. Mostly they were in the range of 23 and 27, but some of them jumped upto 37 while others were as low as 18. Now does these effect my results of are they all right? If there is a problem what may be the possible reason for this? How can I resolve it?
Hi
Housekeeping genes' expression should be consistent otherwise it can't act as a control. I always used a couple of them like beta-actin, GAPDH or 18s. Based on the consistency of the CT we choose the correct one.
As you are getting inconsistent CT for GAPDH, you should check with other housekeeping genes for your experiments.
Best of luck
Farhad
HI Muhammad
Variation like this is not normal. There can be problems with your annealing temperature.
Make sure
1: primers are exon spanning primers
2: Annealing temperature is correct along with the amplicon size and time given for amplification (10 or 15 seconds for 100-150 bp)
3: Try other housekeeping genes, like Succinate dehydrogenase or RPLP0 which are not related to glycolysis which can be easily affected by several factors.
4: I am sure u are taking equal amount of RNA for reverse transcription (1 µg or more), make sure there is no DNA contamination in ur RNA sample. If there is DNA contamination, DNA can contribute to the estimating quantitiy of RNA leading to unequal amount of RNA for cDNA synthesis.
Ct values above 30 or 32 are usually not valid results or indicate absence of cDNA template and the resultant amplification is of primer dimer.
All the best for your results.
Kind regards
Omkar Panchal
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