Recently, we described a technique that allows us to prepare probes for expression profiling from 0.5-1 μg RNA without template or signal amplification. However, we were unable to use this method to study cells harvested by needle biopsy, cell sorting, or laser capture microdissection. Here we give a new protocol for amplifying RNA with multiple reaction cycles and preparing fluorescent probes from approximately 10 cells. We use random 9-mers with a T3 RNA polymerase recognition sequence on the 5′ end for every round of cDNA synthesis except the first. The latter is primed with oligo(dT) with a T7 RNA polymerase recognition sequence on the 5′ end. Results were highly reproducible and reliable, and the products generated using our method seemed comparable to those produced using the RiboAmp RNA kit when both were used to do two cycles of amplification. To test our method's utility, we lysed cells directly into reverse transcription buffer containing RNase inhibitor and performed three rounds of RNA amplification. The expression profiles of mouse C2 and NIH 3T3 cells obtained with 11 232-element arrays using amplified RNAs were similar to those seen when probes were prepared from unamplified templates.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)