Alternative exon-specific PCR method for the analysis of human CD44 isoform expression.
CD44 is a widely expressed cell-surface transmembrane glycoprotein involved in diverse adhesive processes. Its isoforms have been implicated in tumor progression and are considered a promising marker for evaluation of the metastatic potential of various tumors. Several methods have been described for the analysis of CD44 isoforms in tumor cells, including immuno-histochemistry, RT-PCR followed by hybridization and nested RT-PCR. We describe an alternative nested PCR for the analysis of CD44 isoform expression in various malignancies. Total RNA was isolated from various shock-frozen tissues from human tumors, reverse-transcribed and PCR-amplified using CD44-specific primers. Reverse-transcription was performed by two different methods, either using Tth-polymerase or MMLV-RT. Exon-specific amplification was then carried out using specific primers for each variable exon. Amplification products were assayed by agarose gel electrophoresis. Comparison of the patterns obtained from the first amplification and from the exon-specific amplification allowed to identify exons expressed by tumor tissues, as well as the genomic organization of CD44 isoforms. The method developed proved to be sensitive, reliable and inexpensive in comparison with other methods. It can be performed even in solid tumors and for numerous samples, and is suitable for laboratories with limited resources.
Lockhart
M S
MS
IDEHU-CONICET, Catedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Gravisaco
M J
MJ
Mongini
C
C
Waldner
C
C
Alvarez
E
E
Hajos
S E
SE
D001943:Breast Neoplasms / Q000737:chemistry*; D003110:Colonic Neoplasms / Q000737:chemistry*; D005091:Exons / Q000235:genetics*; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis / Q000235:genetics*; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D018960:Hyaluronan Receptors; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D012334:RNA, Neoplasm