What is the suitable product size for conventional RT-PCR analysis - products with size over 1 kb? (Sep/03/2007 )
I have cloned and sequenced a cDNA fragment of 1200 bp. Can i use the same pair of primers for conventional RT-PCR analysis (not real-time RT-PCR). I need to find out the expression pattern of mRNA in different types of tissues. Is the size of amplicons affect the efficiency of RT-PCR like real-time PCR (i always heard that we should use 75-150 bp in real-time PCR for SYGR)? My internal control is only 200 bp. Thanks.
1200bp is too long for regular RT-PCR, the ideal products are about 300-500bp
Ideally, you'd like to use a similar size for your test as you would your control (to ensure similar PCR efficiencies). Personally, I go by the motto of "shorter is better". I like RT-PCR amplicons under 200bp and LOVE those around 100bp. It reduces cycling time, etc.
the techical RTPCR and qRTPCR platform at the institute strongly recommends 100bp, and may go up to 150 if no strict specificity is possible. With more amplification, it's supposed that the amplification is not 100%
Thanks for the comments. Lets say I'm working on two isoform genes which share high similarity in sequence. Should I still design primers which give me short product size like 100-200bp? How can I make sure that the primer are specific for one isoform but not the other isoform? Initially, I thought that longer product is more specific.
Longer primers, not product, are more specific. Align your two isoforms and design your isoform-specific primers in regions where the two isoforms differ, and try to make the 3' terminal bases of your primers isoform specific.
Thank you very much for all the comments. Seems like all the questions that are playing in my mind got their answers.....thanks.
Oops, one more question, since I have the 1200 bp sequence. If i design a pair of gene specific primer from that sequence and I got the desire product size, should i sequence it again?