Hi ALL,
I am using a pair of primers to amplify a region in my gene of interest from cDNA samples. The cDNA samples are extracted from tissues of mouse of different ages. The gene is known to have decerased expression level when mouse ages. However, I did not see any change of the RT-PCR amplicon band intensities on agarose gel, indicating no change for the transcript level. I did not saturate the PCR products as I tried different cycle numbers (from 23 to 30 cycles). What could be the possible reasons? Should I design new primers targeting a different regions in my gene? Thank you for the help!
End-point PCR and agarose gel electrohoresis are not suitable for the detection of changes in gene expression, and visual inspection is not sensitive enough to detect minor differences; nor does it allow any normalization to an internal control.
You need to set-up a quantitative RT-PCR, either using a TaqMan or SYBR approach, and normalize the expression to one or better two appropriate house-keeping genes, to control for minor differences in input and/or tissue-specific expression.
Talk to your supervisors and senior lab members whether you have the necessary equipment and seek their advice how to design the respective RT-qPCR assays.
Sabine Strehl Thank you for the suggestion! My gene of interest shows a great reduction over time in other published work (from high transcript abundance to zero expression), so I would expect that the RT-PCR could reflect the change in transcript abundance even though it may not be as sensitive as qRT-PCR.
I agree with Sabine, you should perform a RT-qPCR and standardize the amount of transcript with a house keeping agent such as tubulin or actin (check the literature to identify the most appropriate ones according to your conditions). To this, I can add and recommend that you check the specificity of your primers, perhaps you are amplifying another gene, that you extract the band from the agarose gel and send it to sequences to prove that the sequence of your gene of interest is correct, or directly design other primers (primers are very cheap). I recommend primerblast https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Good luck
Sequencing amplification products is always a good option to determine the specificity of the PCR. In addition, you should at least run an end-point PCR with a low number of cycles for an appropriate house-keeping gene as control for your input.
Could it be possible that your primers also amplify genomic DNA, which basically always contaminates your RNA unless you conduct a DNase treatment step. If so, design exon-exon spanning primers.
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