I am facing problems like non specific amplification or no amplification in my PCR. Can the RT reaction be optimised using specific RT enzymes? I have tried with MMLV and SS3RT from kits.
The 5' region is often tough to get. That is the reason for developing 5'RACE techniques. A nice variant is the RLM-RACE commercialized by Life Technologies (formerly Ambion).
However if you just need just that stretch of DNA for some purpose and it is already identified you may just amplify it from genomic DNA.
You need to add more information on what you are trying to achieve and how, including a picture of "non specific amplification" and a list of positive controls for the "no amplification".
Broadly speaking, start with the following:
1. Do you know that what your amplicon is actually expressed in your RNA source? Is the gene expressed? Is it the correct splice isoform?
2. If this is just a cloning, is the amplicon on a single exon? If so, why not extract genomic DNA and clone by PCR instead of RT-PCR?
3. Primer your RT using random hexamers, not oligo-dT. The enzyme's processivity is limited, especially if it encounters high-GC stretches forming secondary stretches, so if you start the reverse transcription from the very 3' polyA tail, the RT won't make it to the 5'UTR. Different enzymes will have a negligible effect.
4. Assuming that known primers work well with your RT template, optimise your 5'UTR PCR! Primers, Mg, DMSO, betaine, annealing temperature, extension time, touch down PCR... There is a very long list. Upload information about your amplicon and a picture of a gel so we can help you troubleshoot.
Good luck!
I have never tried SS3RT, but I have tried MMLV in the past and found that it was 10,000-50,000X less productive than Superscript III for cDNA production prior to qPCR/PCR. So, i've been 'burned' by MMLV in the past and will never use it again. Dr. Hall's comment #2 above is extremely important to take notice of. The choice of a more highly thermostable, highly processive RT enzyme is an absolute must here (e.g., Thermo-X Reverse Transcriptase [Life Technologies] has been reported to maintain good activity even up to 70C). I am also in agreement with Dr. Hall's and other's mention of using random hexamers (or nonamers or pentadecamers) for priming (in lieu of oligo d[T]n priming); this should also be a given in your situation if you should choose not to pursue a 5'-RACE method. Best of luck Amrita~!
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