Isolation of mRNA and genomic DNA from epithelial cells in human milk and amplification by PCR.
Studies on the regulation of human milk protein genes and suitable cell culture systems have been limited due to restricted availability of tissue samples from lactating women. Although mammary gland tissue has been available from mammary reduction surgery, studies on tissue-specific expression of milk protein genes require samples obtained during specific stages of expression in the tissue, i.e., lactation. We have therefore developed a technique to isolate mRNA and genomic DNA directly from epithelial cells isolated from human milk and used PCR methodology to specifically amplify the cDNA and genomic DNA for human beta-casein. When comparing fresh human milk with milk that has been stored for five hours at 37 degrees C or frozen and thawed, we found that the amount of mRNA isolated was considerably higher from fresh milk. However, in spite of this, we could still isolate intact mRNA from frozen milk. The described methodology should be useful in studies on milk protein gene expression during lactation and studies on variants of milk protein genes.
Department of Microbiology, University of Umeå, Sweden.
S
S
Lindquist
L
L
Hansson
O
O
Hernell
B
B
Lönnerdal
J
J
Normark
M
M
Strömquist
S
S
Bergström
D001483:Base Sequence; D002364:Caseins / Q000235:genetics*; D004247:DNA / Q000302:isolation & purification*; D004848:Epithelium / Q000737:chemistry; D005260:Female; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000302:isolation & purification*
D002364:Caseins; D012333:RNA, Messenger; D004247:DNA