A lung tissue bank for gene expression studies in chronic obstructive pulmonary disease.
A bank of surgically resected human lung tissues frozen at -70 degrees C after being inflated with support medium for cutting frozen tissue and a separate group inflated with fixative and embedded in paraffin has been established for studies of chronic obstructive pulmonary disease. The present report concerns the quality of RNA that can be extracted from these frozen and fixed tissue samples and from cells obtained from them by laser capture microdissection. The results show that the RNA yield was 257+/-183 ng/mg and 77+/-56 ng/mg from randomly selected frozen and paraffin-embedded tissue, respectively. Intact 18S and 28S rRNA subunits were present in 11/23 frozen and 2/6 paraffin-embedded specimens. The 375-bp actin and 296-bp glyceraldehdye 3-phosphate dehydrogenase targets were amplified by reverse transcription-PCR from both sources and the 983-bp glyceraldehdye 3-phosphate dehydrogenase and 499-bp nonhousekeeping integrin-linked kinase targets from frozen tissue. The minimal amount of RNA required for reverse transcription-PCR of 296-bp glyceraldehdye 3-phosphate dehydrogenase target was 29 pg from frozen tissue when RNA subunits were present and 144 pg when these subunits were absent compared to 0.8 ng from paraffin-embedded tissue. Ten laser pulses were required to laser capture sufficient cells from frozen tissue to detect amplification of the 375-bp actin target while more pulses were required for equivalent amplification from paraffin-embedded tissue. Storage time had no detectable effect on RNA quality. We conclude that both frozen and paraffin-embedded tissues as well as laser-captured cells are suitable for gene expression studies but frozen tissue offered greater sensitivity.
Ding
Lily
L
Department of Pathology, University of British Columbia iCAPTURE Centre/ McDonald Research Laboratories, Vancouver, British Columbia, Canada.
Quinlan
Kevin B
KB
Elliott
W Mark
WM
Hamodat
Mowafak
M
Paré
Peter D
PD
Hogg
James C
JC
Hayashi
Shizu
S
D000199:Actins / Q000235:genetics*; D015139:Blotting, Southern / Q000379:methods; D002460:Cell Line; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006801:Humans; D029424:Pulmonary Disease, Chronic Obstructive / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014015:Tissue Banks
D000199:Actins; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases