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The major concern in handling RNA is the control of ribonuclease activity.
RNases introduced via accidental contamination
RNases liberated by cellular disruption
RNases are very stable.
RNases are extremely active enzymes that do not require co-factors.
RNases are quite difficult to inactivate and small amounts are enough
to destroy RNA.
All plastic and glass containers must be treated to eliminate contamination
by RNases.
RNases arise readily from bacteria and molds present in dust and on
skin and clothing.
Disposable sterile polypropylene tubes are highly recommended as they are generally RNase-free due care taken during manufacture and handling.
Non-disposable plasticware or glassware can be washed with detergent, rinsed several times with sterile distilled water, followed by thorough rinses with 0.1 N NaOH, 1mM EDTA and, finally, RNase-free sterile distilled water.
Alternatively, glassware can be washed with detergent, well rinsed and baked in a dry oven at 240 degrees C for four or more hours. Please note that autoclaving will not completely inactivate all RNases.
Glassware can be treated with DEPC (diethyl pyrocarbonate) by filling glassware with 0.1% DEPC, left to stand 12 at 37C or more hours and then autoclaved to eliminate the DEPC.
Handle DEPC (diethyl pyrocarbonate) very carefully (gloves and fume-hood) as it is a suspected carcinogen.
Electrophoresis tanks are cleaned with detergent, rinse several times with RNase-free water, rinsed with ethanol and allowed to dry.
Aqueous solutions, including water, can be can be treated with 0.1%
DEPC (diethyl pyrocarbonate), left to stand 12 or more hours and then autoclaved.
Notes on DEPC (diethyl pyrocarbonate):
Diethyl pyrocarbonate (DEPC) is a strong inhibitor of most but not
all RNases via a covalent modification reaction.
DEPC cannot be used to directly decontaminate Tris-based buffers as
the DEPC rapidly decomposes into ethanol and carbon dioxide in the presence
of the primary amines of Tris. For Tris-buffers, treat the distilled
water first, then dissolve Tris in the treated water.
Solutions may be autoclaved to eliminate remaining traces of DEPC.
Handle DEPC (diethyl pyrocarbonate) very carefully (gloves and fume-hood)
as it is a suspected carcinogen.
As a general rule, ~30 mg of animal tissue will yield RNA near the 100 ug capacity of the column.
For mouse and rat tissues, the following yields of ug of RNA from 10
mg of tissue have been empirically determined.
Embryo ... 25 ug
Brain ... 8 ug
Heart ... 10 ug
Kidney ... 35 ug
Liver ... 40 ug
Spleen ... 35 ug
Thymus ... 45 ug
All three major classes of RNA, mRNA, tRNA, & rRNA, are synthesized
by transcription of the appropriate genes and are involved in protein synthesis.
1) mRNA carries the message from the DNA to the
ribosome.
2) rRNA are major structural components of the protein-synthesizing
ribosome.
3) tRNA act as adaptor molecules in aligning the
amino acids according to the sequence present in the mRNA.
RNA maintains an hydroxyl group at the 2 prime position of the ribose
sugar, DNA does not.
One strand of the DNA duplex (the template strand) is transcribed into
a segment of mRNA shown, according to the same base-pairing rules used
in DNA replication, except the base U is used in RNA in place of T.
The complementary DNA strand, with a sequence essentially identical
to that of the mRNA, is called the coding strand.
RNA polymerase moves along the template strand of the DNA in the 3 prime to 5 prime direction, and the RNA molecule grows in the 5 prime to 3 prime direction.
Although transcription in eukaryotes is similar to that in prockaryotes,
the process appears to be complex.
Instead of one RNA polymerase, there are three involved in eukaryotic
transcription.
RNA polymerase II produces most mRNAs and snRNAs.
RNA polymerase II: The typical promoter for RNA polymerase II
has a short initiator sequence, consisting mostly of pyrimidines and usually
a TATA box about 25 bases upstream from the startpoint.
This type of promoter (with or without the TATA box) is often called
a polymerase II core promoter, because for most genes a variety of upstream
control elements also play important roles in the initiation of transcription.
General transcription factors and the polymerase undergo a pattern of
sequential binding to initate transcription of nuclear genes.
Termination signals end the transcription of RNA by RNA polymerase
I and RNA polymerase III without the activity of hairpin structures as
seen in prokaryotes.
mRNA is cleaved 10 to 35 base-pairs downstream of a AAUAAA sequence
(which acts as a poly-A tail addition signal).
Messenger RNA in eukaryotes is first made as heterogeneous nuclear mRNA
(or pre-mRNA) then processed into mature mRNA through the addition of a
5 prime cap structure, addition of poly-A tails and the splicing out of
introns.
To give the mRNA stability, a 5 prime "cap" (a guanosine nucleotide
methylated at the 7th position) is joined to the 1st nucleotide in an unusual
"5 prime to 5 prime" linkage (sort of "backwards").
During the capping process, the first two nucleotides of the message
may also become methylated
Transcription of eukaryotic pre-mRNAs often proceeds beyond the 3prime
end of the mature mRNA.
An AAUAAA sequence located slightly upstream from the proper 3prime
end then signals that the RNA chain should be cleaved about 10-35 nucleotides
downstream from the signal site, followed by addition of a poly-A tail
catalyzed by poly(A) polymerase.
The spliceosome is an RNA-protein complex that splices intron-containing pre-mRNA in the eukaryotic nucleus.
Most mRNA molecules have a high turn over rate as the molecules are
rapidly degraded and replaced while tRNAs and rRNAs are relatively stable.
The half-lives of eukaryotic mRNA range from minutes to days.
Transcription allows amplification of the genetic information because
many copies of the mRNA can be produced to direct a great deal of protein
synthesis.
Recombinant DNA molecules are produced by ...
1) cleaving DNA from two different sources with
restriction endonucleases (restriction enzymes),
2) mixing the fragments together to allow the ends
of the fragments to interact and
3) linking the fragments with DNA ligase.
The cloning of specific DNA fragments usually
involve:
1) Insertion of DNA into a vector (a recombinant
vector)
2) Introduction of recombinant vector into cells
(usually E. coli)
3) Amplification of recombinant vector in the cells
4) Selection of cells that carry the recombinant
vector.
5) Identification of correct recombinant clone.
Often a "shotgun" approach is used to produce clones.
This means that instead of starting with a known specific fragment
of DNA, "all" the DNA from a source (as relatively random pieces
is cloned into a vector) to result in a library of clones.
If the source of the DNA is the genome of an organism, then the library
is refered to as a genomic library.
To examine the expressed genes of an organism, the mRNA can be "converted"
into a complementary DNA (cDNA) library through
the use of the enzyme reverse transcriptase.
cDNA is made by annealing poly-T primers to the poly-A tails of isolated
mRNA and synthesizing ssDNA from the mRNA template with reverse transciptase.
The RNA is hydrolysed and a DNA polyermerase generates the second strand
to make dsDNA.
The cDNA is then inserted into a vector and propagated as above.
email me at bestave@mun.ca