RT-PCR product smaller than expected

[Joey]

Hi. I'm very new to molecular biology and am having trouble with a particular gene expression.

My first set of primers did not give me any product and my second set have given me a product approx half the size I was expecting - Should have been 657bp product, I got ~300bp product. I have done a temp gradient and all products were the same size and seemed specific though faint. Is it worth getting the 300bp product sequenced or should I try another set of primers?

[mchlbrmn]

We can't tell, perhaps it's some earthshattering alternate splice that will cure cancer or lead to a better spandex bra, or something. If all the temperatures used gave equal amounts of product, you haven't gone up as high as you can. Perhaps higher anneal temp, lower Mg++ (down to 1.5 or 1 mM) cosolvent (5-10% DMSO, or something, if you have the energy) will clean up the PCR. Or, perhaps the trancript is a size you didn't predict. Also, you can try combinations of the forward and reverse primers in your 2 primer sets to get 2 new sets, unless they're not compatatble becaust the ends will complement to form dimers, or there's drastic general complementarity.

For your first set, did you lower the anneal temp? The predicted temp is only a guidline and different methods give temps varying by 10 degrees or more, sometimes.

[relaxin]

It looks like your PCR product is a product of mispriming or your RNA is not good (either it is degraded or is not a good source for the gene you are interested). If your positive control works, then your RNA is not degraded.
Before you rush to get another set of primers, you need check with someone on the design of primers, and make sure that they are within the guidelines of primer design. Otherwise, you will face further frustration.