Isolation, detection, and amplification of intact mRNA from dermatome strips, epidermal sheets, and sorted epidermal cells.
Three different strategies for isolating RNA from epidermal cells were compared. Starting with dermatome sections frozen or disaggregated epidermal cells purified by fluorescence activated cell sorting (FACS), RNA was isolated with a guanidinium thiocyanate technique. Specific mRNA were detected by Northern blot analysis (involucrin, keratin 5, actin), or by reverse transcription and amplification with the polymerase chain reaction (PCR), using primers specific for keratinocyte products (keratins 1 and 14) and Langerhans cells (CD1a). Messenger RNA's characteristic of Langerhans cells and of keratinocytes at different stages of differentiation were detected in dermatome and epidermal sheet preparations as well as in FACS-separated cells. The use of snap-frozen dermatome sections allows the isolation of RNA from epidermis that has undergone minimal trauma and is very close to its in vivo state, but that includes RNA from some dermal cells. Extraction of RNA from Dispase-separated sheets involves slightly more manipulation of the epidermis but provides a sample free from dermal contaminants. PCR analysis of sorted epidermal cells is both sensitive and specific, but involves still greater manipulation. This final technique, however, allows the investigation of mRNA produced by small groups of epidermal cells that are still much closer to their in vivo state than if they had been cultured. By combining these techniques it is possible to determine the baseline production of specific mRNA in the skin in vivo and to assign their production to specific groups of cells with a sensitivity and specificity greater than any approach previously described.
Longley
J
J
Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510.
Ding
T G
TG
Cuono
C
C
Durden
F
F
Crooks
C
C
Hufeisen
S
S
Eckert
R
R
Wood
G S
GS
D001483:Base Sequence; D015152:Blotting, Northern; D010450:Endopeptidases; D004817:Epidermis / Q000033:anatomy & histology* / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods; D005784:Gene Amplification; D006801:Humans; D008867:Microtomy / Q000379:methods; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*
D012333:RNA, Messenger; D010450:Endopeptidases; C023139:dispase