should I use a higher concentration RNA and cDNA or increase the number of PCR cycles to be more than 42?
Try nested pcr reamplification of the first round pcr product with primers internal to the first round outer primers
Short version: If you don't see it after 42 cycles, you won't see it after more. Increase the amount of template.
Long version: The best move here depends on what you are trying to do. Is this quantitative RT-PCR, or do you need to amplify the cDNA for cloning etc.?
If you're trying to do qPCR: 42 cycles is a mathematical limit. If even one copy of your template existed in the reaction tube, you would see it above limit of detection after 42 cycles, so increasing the cycle count is futile (any amplification you see after 43+ artifactual - primer dimer, probe degradation, etc) Instead, check these first:
- Do you know the template is good? (Do reference gene primers amplify from this template after a reasonable number of cycles - 25-30? If not, try your primers with known-good template.)
- Do you know the primers work? (Do they amplify from a positive control template in which the gene is definitely expressed, with good efficiency and kinetics? If not, design new primers.)
- Are reaction conditions optimised? (Check annealing temp in particular)
- Are your instrument settings correct? (Is your instrument imaging in the correct channel, during the extension phase of the PCR reaction? Run your samples on a gel, if you see a band even though the instrument reports no amplification, the problem is with your instrument or settings.)
- If all of the above is good, try reactions with considerably more template. When I was dealing with low-expression genes I used: 500-1000ng of RNA per 20uL RT reaction, diluted cDNA to 40uL with water, used 1uL of this template per qPCR.
If you're trying to do cloning - increasing the amount of template is the first thing I would try. If you're seeing literally nothing after 42 cycles it will need to be a big increase, maybe 100-fold. If that doesn't work, check these:
- Is the extension step of your PCR reaction long enough to amplify the full length cDNA? If it's especially long (more than about 2-5kb) the default time may not be long enough.
- Did your RT work? Use known-good primers to check that you can amplify other genes from the same template.
- Was the RNA degraded? You need high quality RNA input to get long PCR products.
- How was the reverse transcription primed? Poly-T or gene specific priming might be necessary to get long products because sequential annealing of random primers on the same transcript can cause the RTase to fall off, resulting in lots of short random products but almost no full-length cDNA.
- Is the template difficult? Try playing with the annealing temperature. If the template GC content is high or contains secondary structures, consider trying a PCR master mix optimised for high GC or adding enhancers (betaine or DMSO) to the PCR reaction.
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