I have isolated RNA with high purity and concentration from banana and converted it to cDNA then amplified with reference gene (Actin, tubulin, ubiquitin) and specific primer (no cross dimer, hair pin, GC clamp and long runs is ok, only have a self dimer), but not appear on electrophoresis gel even for reference gene.
I have re-isolated and remade cDNA for two batch and re-amplified, but the result is still bad.
How if i use that cDNA for qPCR to make sure that the reaction is work? Is that will be ok?
Maybe you should amplify the higher concentration of DNA in order to detect the bands on an agarose gel or you might have forgotten to add a sample loading dye.
Even if you don't see any bands on the gel, you should still run a q-PCR. If your samples have a relatively high Ct-value, then, it means that you have used very little DNA and vice versa. Therefore, you can use the results of your q-PCR to determine where the problem arises.
Hope I answered your question.
In primer design, self-dimer is of concern and can affect your amplification. But, even the reference gene has not got amplified. So, first run the control to ensure that the template is pure enough. Presume, you loaded atleast 5 to 10 ul of the amplified product in which case you should see the band on gel. If sample dye is not added to the sample, then you cant load the sample itself as it will spread out and disperse.
Dear Lulu,
How bad is your self-dimer - I assume this is only one set of primers? I assume that as you are using three reference genes, you're not getting amplification in any of them?
That would point to a problem before the PCR step, so:
How did you determine that you have RNA with high purity and concentration? Spectrophotometric methods do not tell you anything about the "quality" of RNA, ie if your ribonucleotides are actually still part of continuous sequences or are simply chopped up pieces. You can get perfectly nice 260/280 ratios and high concentrations but have only pieces of RNA in there.
How much RNA did you use in your reverse transcriptase reaction? Are you using gene specific primers in the RT step or oligo-dT or random hexamers? Is the cDNA sequence high in G/C content (Superscript III is meant to be good for high G/C content sequences)? What kit are you using? Have you tried a new kit (in case your current one has non-functional enzyme)?
The primers for the PCR - I assume that you have used the banana sequence to design them? Again, have you tried a new lot of Polymerase and nucleotides?
Hope that will get you thinking...
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