I did the RNA extraction from mice's liver three times over, and the concentration is around 1000ng/ul.(260/280 is 1.9-2.1),then i used 500ng to make cDNA, but when i use taqman probes to do the qPCR, i can not get the results, the 90% samples 'CT always showed "undetermined",even the Taq (vic) probe can not be detected.Can an experienced scientist tell me what went wrong? i'd be grateful if you could answer my question.
What do your controls look like? You should have a full set including: water (no template) for each probe; a no-RT control with gDNA for each probe; a known housekeeping gene for each of your samples AND a known positive control for both the housekeeping gene and the gene-of-interest. Plus your standard curve.
As Katie A Burnette mentioned above , you should describe your issues more clearly with images if possible. In the meantime, I suggest to use one-step PCR kit instead of two-steps separately. By doing so, you can eliminate the error in the reverse transcription step, and you also may know if anything was wrong with the reverse transcription.
Hi, I agree with the suggestions above. In addition, I will suggest to do initial optimizations on regular PCR to see if your cDNA synthesis and transcript level of gene of interest is fine or not, and then shift to qPCR analysis.
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