Differential display reverse transcription-polymerase chain reaction to identify novel biomolecules in arthritis research.
Differential display is one of the simplest techniques for discovering novel transcripts when comparing gene expression in biological systems. The method can be carried out on small amounts of total RNA and permits the simultaneous comparison of multiple independent samples in a single experiment. The methodology is versatile in that it allows the researcher to adapt the existing protocol by varying the selection of oligonucleotide primers used in the PCR. The putative differentials, which are isolated from the polyacrylamide gels as cDNA fragments of approx 100 to 500 bases in size, can be instantly recognized after sequencing by searching the nucleotide databases. However, one of the drawbacks is the isolation of false positives and hence the need to confirm the results of the screen by another method. Once the true differentials have been identified, further downstream work is also required to recognize which splice variant of the transcript gives rise to the expression differences and whether the gene expression results are corroborated at the protein level.
Ali
Manir
M
Leeds Institute of Molecular Medicine, University of Leeds, St. James's University Hospital, UK.
Isaacs
John D
JD
D001168:Arthritis / Q000235:genetics* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism*; D030561:Databases, Nucleic Acid; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D015415:Biomarkers; D012313:RNA