Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.
Leethanakul
C
C
Oral & Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Building 30, Room 212, Bethesda, MD 20892-4330, USA.
Patel
V
V
Gillespie
J
J
Shillitoe
E
E
Kellman
R M
RM
Ensley
J F
JF
Limwongse
V
V
Emmert-Buck
M R
MR
Krizman
D B
DB
Gutkind
J S
JS
D000368:Aged; D001706:Biopsy / Q000379:methods; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D016022:Case-Control Studies; D018076:DNA, Complementary / Q000235:genetics*; D004210:Dissection / Q000295:instrumentation / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D007834:Lasers; D008297:Male; D008875:Middle Aged; D009061:Mouth Mucosa / Q000473:pathology; D009062:Mouth Neoplasms / Q000235:genetics* / Q000473:pathology; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012334:RNA, Neoplasm