Reverse transcription-polymerase chain reaction on a microarray: the integrating concept of "active arrays".
In this report we describe the proof of principle of a reverse transcription polymerase chain reaction (RT-PCR) but on-chip, with immobilized specific primers using a transcriptome of mouse-muscle fibroblasts for detection of muscle-specific expression products of these cells. The isolated total mRNA was directly incubated on an array of immobilized and solubilized specific primers, which allow the amplification of certain muscle-specific RNAs via its immobilized cDNAs. In contrast to others, the immobilized cDNA-products were directly synthesized on the chip by applying covalently bound specific primers. The products were detected by the incorporated and fluorophore-modified specific primers of the subsequently synthezised second strand. In addition, this second-strand served as a further template (like the basically used mRNA) in the subsequent solid-phase-PCR to amplify first-strand cDNA copies at the remaining immobilized specific primer-probes. This is the intrinsic factor of the amplification of certain signals of this application. The specific cDNA templates of genes coding for subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm), and ATPase (Atp2a2) were amplified on a biochip by RT-PCR directly from freshly isolated mRNA. The resulting procedure allows the detection of mRNA sequences from less than 5 pg of total RNA preparations.
von Nickisch-Rosenegk
Markus
M
Fraunhofer Institute for Biomedical Engineering (IBMT), Branch Potsdam, Department Nanobiotechnology and Nanomedicine, Am Mühlenberg 13, 14476, Potsdam-Golm, Germany. markus.nickisch@ibmt.fraunhofer.de
Marschan
Xenia
X
Andresen
Dennie
D
Bier
Frank F
FF
D000109:Acetylcholine / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D052283:Creatine Kinase, MM Form / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D005815:Genetic Code; D051379:Mice; D046228:Microarray Analysis / Q000379:methods*; D018008:Myogenin / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D018076:DNA, Complementary; D018008:Myogenin; D012333:RNA, Messenger; D052283:Creatine Kinase, MM Form; D000251:Adenosine Triphosphatases; D000109:Acetylcholine