Inactivation of tumor suppressor and metastasis suppressor genes via epigenetic silencing is a frequent event in human cancers. Recent work has shown new mechanisms of epigenetic silencing, based on the occurrence of long noncoding promoter-spanning antisense and/or sense RNAs (lncRNAs), which constitute part of chromatin silencing complexes. Using reverse transcription polymerase chain reaction (RT-PCR), we have started to scan "triple negative" and Her2-overexpressing breast cancer cell lines for directional/bidirectional transcription through promoters of tumor suppressor and metastasis suppressor genes known to be epigenetically silenced in vivo. Surprisingly, we found that RT-PCR-amplified products were obtained at high frequency in the absence of exogenous primers. These amplified products resulted from RT priming via transcripts originating from promoter or upstream spanning regions. Consequently, this priming overruled directionality determination and led to false detection-identification of such lncRNAs. We show that this prevalent "no primer" artifact can be eliminated by treating the RNA preparations with periodate, performing RT reactions at highly elevated temperatures, or a combination of both. These experimental improvements enabled determination of the presence and directionality of individual promoter-spanning long noncoding RNAs with certainty. Examples for the BRMS1 metastasis suppressor gene, as well as RAR-β2 and CST6 human tumor suppressor genes, in breast carcinoma cell lines are presented.
|Number of pages||7|
|State||Published - 2013|
Bibliographical noteFunding Information:
We thank J. Price for a biological reagent and G. Kaufmann for his thoughtful suggestion in treating RNA preparations with periodate. This work was supported by a grant from the David Orgler Fund for Cancer Research to Dan Canaani. Sivan Tzadok was supported by a TAU Rector fellowship. Yafit Hachmo was supported by a van Baytes Cancer Research fellowship.
- Epigenetic gene silencing
- Long noncoding RNAs