title: Directionality of noncoding human RNAs: how to avoid artifacts.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Sivan
authors.initials: S
authors.lastname: Tzadok
authors.forename: Yarden
authors.initials: Y
authors.lastname: Caspin
authors.forename: Yafit
authors.initials: Y
authors.lastname: Hachmo
authors.forename: Dan
authors.initials: D
authors.lastname: Canaani
authors.forename: Iris
authors.initials: I
authors.lastname: Dotan
mesh_terms: D016477:Artifacts; D045744:Cell Line, Tumor; D055332:Cystatin M / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics; D022661:RNA, Untranslated / Q000235:genetics*; D018168:Receptors, Retinoic Acid / Q000235:genetics; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: C410024:BRMS1 protein, human; C525104:CST6 protein, human; D055332:Cystatin M; D017931:DNA Primers; D009363:Neoplasm Proteins; D016372:RNA, Antisense; D022661:RNA, Untranslated; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; C082925:retinoic acid receptor beta