Biomedical Engineering Reference
In-Depth Information
29. UV light box, camera for visualizing EtBr-stained agarose gels.
30. Scalpel with disposable blades.
31. Primers:
Primer sets can be ordered commercially (Genomyx or GenHunter) or custom-
ordered. A set of custom primers might include:
One-base anchored primers:
GGGCG AAGCT TTTTT TTTTT A
GGGCG AAGCT TTTTT TTTTT C
GGGCG AAGCT TTTTT TTTTT G
Upstream “random” primers of the form:
GACAC TATAG AATTC
NNNNN NN
, where
N
is any base.
(As a starting point, try GTTGTGC, AACGAGG, TCTCTGG, and
TGGTCAG)
Reamplifi cation primers:
Downstream: CGCGC GTAAT ACGAC TCACT ATAGG GCGAA GCTTT
TTTTT TTT
Upstream: CATAC GATTT AGGTG ACACT ATAGA ATTC
(These reamplifi cation primers complete the T7, SP6 RNA polymerase sites
on either side of the reamplifi ed DDPCR fragment).
3. Methods
3.1. RNA Extraction
Total RNA can be extracted using any of a variety of standard methods. It is
not necessary to extract further only the poly(A)
+
(mRNA) component, but it is
important to obtain high-quality (i.e., undegraded) RNA. The RNA should also
be treated with RNase-free DNase I to remove any DNA contamination (
see
Notes 3
and
4
). Run 0.5 or 1
g of RNA per sample in a simple (nondenaturing)
agarose/ethidium bromide gel to confi rm that the RNA is undegraded (
Fig. 2
).
This gel also confi rms that the relative amounts of RNA in each sample are
even (accurate quantitation), and should be performed immediately before the
reverse transcription reactions.
µ
3.2. RT Reactions
(Total of 20
µ
L; can be scaled up or down by a factor of 2.)
1. Assemble all components on ice (
see
Notes 5
and
6
).
RT Mix:
(per reaction)
(example: 27 reactions-worth)
2
O
6.9
µ
L
186.3
µ
L
.3
5X RT buffer
4.0
µ
L
108
µ
L
.3
dNTP (250
µ
M
)
1.6
µ
L
43.2
µ
L
DTT (0.1
M
)
2.0
µ
L
1
54
µ
L
.3
RNasin
0.5
µ
L
13.5
µ
L
Total
15
µ
L
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