Role of normalization in the elimination of abundant myelin sequences in spinal cord cDNA libraries produced by suppression subtractive hybridization.
Spinal cord libraries subtracted against visual cortex using suppression subtractive hybridization SSH are dominated by abundant gene sequences derived from myelin elements. We compared our subtracted library results of three of these abundant sequences to published expressed sequence tag libraries that are not normalized and not subtracted and presumed representatives of murine spinal cord mRNA abundance. We show that: all three abundant sequences, myelin basic protein (Mbp), proteolipid protein (Plp1) and Ferretin heavy chain (Fth1) are highly expressed in spinal cord when this structure is compared to visual cortex; myelin basic protein is represented in our subtracted libraries but at a low frequency, whereas Plp1 and Fth1 represent nearly one-third of all sequences in these libraries; mirror orientation selection, a procedure designed to reduce background sequences, generates libraries very similar in abundance to SSH; proteolipid protein can be reduced in these libraries by adding Plp1 sequences to the driver in the SSH procedure and also by subtracting Plp1 directly from tester and driver. We conclude that adequate normalization is essential to reduce the presence of abundant sequences in SSH libraries.
Solomon Park Research Institute, Kirkland, WA, USA.
K B
KB
Lathia
Z
Z
Yan
P A
PA
Clapshaw
D000818:Animals; D001052:Apoferritins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D020224:Expressed Sequence Tags; D005786:Gene Expression Regulation; D015723:Gene Library; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D004676:Myelin Basic Protein / Q000235:genetics / Q000378:metabolism; D018991:Myelin Proteolipid Protein / Q000235:genetics / Q000378:metabolism; D009186:Myelin Sheath / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000378:metabolism*; D014793:Visual Cortex / Q000378:metabolism
D004676:Myelin Basic Protein; D018991:Myelin Proteolipid Protein; C047516:Plp1 protein, mouse; D012333:RNA, Messenger; D001052:Apoferritins