Stellaris® RNA Fluorescence In Situ Hybridization for the Simultaneous Detection of Immature and Mature Long Noncoding RNAs in Adherent Cells.

RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the detection and localization of RNA, is becoming an increasingly important complement to other gene expression analysis methods. Especially important for long noncoding RNAs (lncRNAs), RNA FISH adds the ability to distinguish between primary and mature lncRNA transcripts and thus to segregate the site of synthesis from the site of action.We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple primary and mature mRNA and lncRNA gene products and RNA variants in fixed mammalian cells. The technique makes use of fluorescently pre-labeled, short DNA oligonucleotides (circa 20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in fluorescent signals that reveal clusters of RNAs or single RNA molecules as punctate spots without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the counting of single transcripts down to one copy per cell. Additionally, by using probe sets with spectrally distinct fluorophores, multiplex analysis of gene-specific RNAs, or RNA variants, can be achieved. The presented examples illustrate how this method can add temporospatial information between the transcription event and both the location and the endurance of the mature lncRNA. We also briefly discuss post-processing of images and spot counting to demonstrate the capabilities of this method for the statistical analysis of RNA molecules per cell. This information can be utilized to determine both overall gene expression levels and cell-to-cell gene expression variation.

[1]  K. Morris,et al.  Expanding the functional role of long noncoding RNAs , 2014, Cell Research.

[2]  Mitchell Guttman,et al.  RNA and dynamic nuclear organization , 2014, Science.

[3]  H. E. Johansson,et al.  Stellaris[trade] fluorescence in situ hybridization (FISH) probes: a powerful tool for mRNA detection , 2011 .

[4]  Igor Jurisica,et al.  The c-Myc oncogene directly induces the H19 noncoding RNA by allele-specific binding to potentiate tumorigenesis. , 2006, Cancer research.

[5]  G. Yehia,et al.  Analysis of alterative cleavage and polyadenylation by 3′ region extraction and deep sequencing , 2012, Nature Methods.

[6]  T. Orfeo,et al.  One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. , 1977, Journal of the National Cancer Institute.

[7]  David R. Kelley,et al.  Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre , 2014, Nature Structural &Molecular Biology.

[8]  Brian Munsky,et al.  Transcription Factors Modulate c-Fos Transcriptional Bursts , 2014, Cell reports.

[9]  V. Buckle,et al.  Detection of nascent RNA transcripts by fluorescence in situ hybridization. , 2010, Methods in molecular biology.

[10]  T. Babak,et al.  A quantitative atlas of polyadenylation in five mammals , 2012, Genome research.

[11]  D. Largaespada,et al.  PVT1 dependence in cancer with MYC copy-number increase , 2014, Nature.

[12]  F. Grosveld,et al.  Chromatin interaction mechanism of transcriptional control in vivo , 1998, The EMBO journal.

[13]  David G. Knowles,et al.  Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs , 2012, Genome research.

[14]  Marshall J. Levesque,et al.  Turbo FISH: A Method for Rapid Single Molecule RNA FISH , 2013, PloS one.

[15]  J. Mattick,et al.  The dark matter rises: the expanding world of regulatory RNAs. , 2013, Essays in biochemistry.

[16]  J. Rinn,et al.  linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis. , 2013, Genes & development.

[17]  S. Tilghman,et al.  Locus unlinked to alpha-fetoprotein under the control of the murine raf and Rif genes. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Ohno,et al.  Allele‐specific detection of nascent transcripts by fluorescence in situ hybridization reveals temporal and culture‐induced changes in Igf2 imprinting during pre‐implantation mouse development , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

[19]  Dexing Zhong,et al.  Genome-wide identification and predictive modeling of lincRNAs polyadenylation in cancer genome , 2014, Comput. Biol. Chem..

[20]  E. Dees,et al.  The product of the H19 gene may function as an RNA , 1990, Molecular and cellular biology.

[21]  Marshall J. Levesque,et al.  Visualizing SNVs to quantify allele-specific expression in single cells , 2013, Nature Methods.

[22]  Marshall J. Levesque,et al.  Single chromosome transcriptional profiling reveals chromosome-level regulation of gene expression , 2013, Nature Methods.

[23]  Alfred Wittinghofer,et al.  C2 domains as protein-protein interaction modules in the ciliary transition zone. , 2014, Cell reports.

[24]  A. Hochberg,et al.  Parental imprinting of the human H19 gene , 1992, FEBS letters.

[25]  David L. Spector,et al.  3′ End Processing of a Long Nuclear-Retained Noncoding RNA Yields a tRNA-like Cytoplasmic RNA , 2008, Cell.

[26]  W. Nie,et al.  Characterization of two human lung adenocarcinoma cell lines by reciprocal chromosome painting. , 2010, Dong wu xue yan jiu = Zoological research.

[27]  J. Mcneil,et al.  XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure , 1996, The Journal of cell biology.

[28]  M. Mhlanga,et al.  Chromosomal Contact Permits Transcription between Coregulated Genes , 2013, Cell.

[29]  Data production leads,et al.  An integrated encyclopedia of DNA elements in the human genome , 2012 .

[30]  Xin Wu,et al.  The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods. , 2010, Methods.

[31]  Howard Y. Chang,et al.  Genome regulation by long noncoding RNAs. , 2012, Annual review of biochemistry.

[32]  J. Davis.  Basic cell culture , 2002 .

[33]  Jingyi Fei,et al.  RNA fluorescence in situ hybridization in cultured mammalian cells. , 2015, Methods in molecular biology.

[34]  H. E. Johansson,et al.  Simultaneous detection of nuclear and cytoplasmic RNA variants utilizing Stellaris® RNA fluorescence in situ hybridization in adherent cells. , 2014, Methods in molecular biology.

[35]  P. Cook,et al.  Transcription factories: genome organization and gene regulation. , 2013, Chemical reviews.

[36]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.