RT-PCR troubleshooting

Roland Hübner rhubner at gins.uia.ac.be
Thu Jun 3 02:59:16 EST 1999


 > s.roberts-thomson at PHARMACY.UQ.EDU.AU (Sarah Roberts-Thomson) wrote:
 >
 >I am trying to amplify a particular mRNA target and having little
 >success, although other targets cause us no problems. I know the mRNA is
 >present in the sample RNA we are using. We are using a one step RT-PCR
 >kit and can show that the PCR step is working with our primers by using
 >a brain cDNA library. We also know that all the components in this kit
 >are okay as we can use it to amplify other targets. Does anyone have any
 >suggestions? We have tried a range of magnesium conc, annealing temp etc
 >etc.

Hello,

 could it be that you are trying to pull out a particular neuropeptide
rcpt. mRNA from peripheral nervous system or something similar? 

 Then, consider that you might have way fewer cells in total tissue sample
that express it as compared to brain... Try something like 250 (or more!)
ng poly-A+ mRNA/rxn.

 In case you have alternative splicing going on, perhaps you could get
your experiments to work by adapting the rxn's for a "long" target...

 cDNA libraries (I know for sure about one commercial brain lib) are quite
often found to be "contaminated" by DNA; if you made your target RNA
maximally DNA-free and by bad luck one primer anneals to an intron or
junction, you could miss it even if amplified OK from lib...

Good luck,
 Roland



More information about the Methods mailing list