发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: questions about DNA and hybridization,thanks a lot~
发信站: The unknown SPACE (Tue Apr 23 13:01:21 2002), 站内信件

For such microarray analysis, typically the mRNA is reverse-
transcribed into complementary DNA (cDNA) and labeled with
fluorescence dye or radioisotope. and the single-stranded
cDNA is hybridized to the complementary strand that is already
spread on the matrix with known position. i have only used
microarray from Clontech. theoretically, the RT enzyme with
gene-specific primers can reverse-transcribe the mRNA into
same amount of cDNA. otherwise, you cannot make the comparison
quantitatively.
for your experiment, you try to compare the expression profile
of certain tumor cell with a physiologically normal control.
so you can use two dyes with different colors and label the
cDNA, for example, one red and one green. if you mix equal
amount of labeled cDNA and hybridize to the array, they have
same affinity for hybridization. thus, if they are expressed
at the same level, then equal amount of cDNA will give same
intensity of fluorescence and it can be recognized as yellow
(red+green). however, if certain genes in one type of cells
is upregulated, which means that it is transcribed at higher
level, then the color will show up. downregulation means the
transcripts are less than the other counterpart. the most
important thing is how to normalize and interpret the data.
with global gene normalization, it may be more approapriate.
however, ppl sometimes use "house-keeping" genes such as
tubulin, ribosomal RNA, metabolic enzymes for such purpose,
hypothesizing that expression levels of such RNA are not
changed. statistical models are needed to validate such analysis
and further confirmed by RTPCR/northern blot.

【 在 lustre (紫色的花) 的大作中提到: 】
: You are really of great help and sound very professional:)
: I think I got the part about genome.My question about hybridization is:
: there are two RNA samples,one from for example tumor tissure,and the other is
: reference.
: Now,they are equally mixed and hybridized to the cDNA probes on the chip.As I
: understand,if they are all the complementary part of the cDNA,every one should
: base pair.but why at some spot,tumor RNA did more,which means the gene is
: expressed,does this means tumor RNA becomes more active?What about the less
: tumor RNA ones?Upregulated means some happened,does downregulated means the
: opposite happening?And what is the opposite?The gene shrinks?
: And what is the connection between mRNA and cDNA?
: Really appreciate your help and patience to my weird questions!
: 【 在 leohawk (leohawk) 的大作中提到: 】
: : genome made of several long DNA moleculars, in eukaryotes, several linear
: : one(human has 46, 23 pair, one from mom, one from dad). a linear molecular
: : is a chromosome. in prokaryotes, usually a circular one.
: : gene is a stretch of the DNA molecular
: hybridized
: more
: : DNA usually exist in double helix, based on affinity, hybridizatin is to
: : speciically find the other part (or, those with complementary sequences).
: : more hybridization means stronger signal...if they all hybridize, how can
: : you tell the specificity
: there
: : se two situation?


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