PMID- 26054766
OWN - NLM
STAT- MEDLINE
DCOM- 20160303
LR  - 20181113
IS  - 1940-9818 (Electronic)
IS  - 0736-6205 (Print)
IS  - 0736-6205 (Linking)
VI  - 58
IP  - 6
DP  - 2015 Jun
TI  - Combined in vitro transcription and reverse transcription to amplify and label 
      complex synthetic oligonucleotide probe libraries.
PG  - 301-7
LID - 10.2144/000114298 [doi]
AB  - Oligonucleotide microarrays allow the production of complex custom 
      oligonucleotide libraries for nucleic acid detection-based applications such as 
      fluorescence in situ hybridization (FISH). We have developed a PCR-free method to 
      make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA 
      library. A double-stranded oligonucleotide library is amplified by transcription 
      to create an RNA library. Next, dye- or hapten-conjugate primers are used to 
      reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA 
      is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent 
      probes library. Starting from unique oligonucleotide library constructs, we 
      present two methods to produce single-stranded probe libraries. The two methods 
      differ in the type of reverse transcription (RT) primer, the incorporation of 
      fluorescent dye, and the purification of fluorescent probes. The first method 
      employs dye-labeled reverse transcription primers to produce multiple 
      differentially single-labeled probe subsets from one microarray library. The 
      fluorescent probes are purified from excess primers by oligonucleotide-bead 
      capture. The second method uses an RNA:DNA chimeric primer and amino-modified 
      nucleotides to produce amino-allyl probes. The excess primers and RNA are 
      hydrolyzed under alkaline conditions, followed by probe purification and labeling 
      with amino-reactive dyes. The fluorescent probes created by the combination of 
      transcription and reverse transcription can be used for FISH and to detect any 
      RNA and DNA targets via hybridization.
FAU - Murgha, Yusuf
AU  - Murgha Y
AD  - MYcroarray, Ann Arbor, MI.
FAU - Beliveau, Brian
AU  - Beliveau B
AD  - Department of Genetics, Harvard Medical School, Boston, MA.
FAU - Semrau, Kassandra
AU  - Semrau K
AD  - MYcroarray, Ann Arbor, MI.
FAU - Schwartz, Donald
AU  - Schwartz D
AD  - MYcroarray, Ann Arbor, MI.
FAU - Wu, Chao-Ting
AU  - Wu CT
AD  - Department of Genetics, Harvard Medical School, Boston, MA.
FAU - Gulari, Erdogan
AU  - Gulari E
AD  - MYcroarray, Ann Arbor, MI.
AD  - Department of Chemical Engineering, University of Michigan, Ann Arbor, MI.
FAU - Rouillard, Jean-Marie
AU  - Rouillard JM
AD  - MYcroarray, Ann Arbor, MI.
LA  - eng
GR  - DP1 GM106412/GM/NIGMS NIH HHS/United States
GR  - R43 GM093579/GM/NIGMS NIH HHS/United States
GR  - 1R43GM093579/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20150601
PL  - England
TA  - Biotechniques
JT  - BioTechniques
JID - 8306785
RN  - 0 (DNA Primers)
RN  - 0 (DNA, Single-Stranded)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Oligonucleotide Probes)
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - Base Sequence
MH  - Cell Line
MH  - DNA Primers/chemistry/genetics
MH  - DNA, Single-Stranded/chemistry/*genetics
MH  - Fluorescent Dyes/chemistry/*metabolism
MH  - *Gene Library
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Oligonucleotide Array Sequence Analysis/methods
MH  - Oligonucleotide Probes/chemistry/*genetics
MH  - RNA/chemistry/*genetics
MH  - *Reverse Transcription
MH  - Transcription, Genetic
PMC - PMC4949592
MID - NIHMS798109
OTO - NOTNLM
OT  - Oligopaint FISH probes
OT  - fluorescent in situ hybridization (FISH)
OT  - in vitro transcription (IVT)
OT  - microarrays
OT  - oligonucleotide library pools
OT  - reverse transcription (RT)
OT  - single-stranded DNA (ssDNA) libraries
EDAT- 2014/01/01 00:00
MHDA- 2016/03/05 06:00
PMCR- 2016/07/19
CRDT- 2015/06/10 06:00
PHST- 2014/11/06 00:00 [received]
PHST- 2015/03/26 00:00 [accepted]
PHST- 2015/06/10 06:00 [entrez]
PHST- 2014/01/01 00:00 [pubmed]
PHST- 2016/03/05 06:00 [medline]
PHST- 2016/07/19 00:00 [pmc-release]
AID - 000114298 [pii]
AID - 10.2144/000114298 [doi]
PST - epublish
SO  - Biotechniques. 2015 Jun 1;58(6):301-7. doi: 10.2144/000114298. eCollection 2015 
      Jun.