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I performed the RNA isolation from cell lisate in Trizol with a spin column kit. Prior to RNA isolation, the samples were stored at -80oC. Reading at Nanodrop is ok. RNA gels show both RNA bands. However, after performing the reverse transcription (Qiagen kit), we are not able to obtain any amplification of RPL13 gene throught qPCR using Taqman Mix (Applied Biosystems). The primers and probe are ok. We just can’t figure out the possible existing problems.
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Do you have a positive control for the reverse transcription step? Do you have another PCR primer set that you can use on the cDNA (a house keeping gene) that you can use to check that cDNA is OK. Have you tried spiking in DNA to make sure there are no inhibitors present?
Daniel Tillett
Nucleics Support
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