Biomedical Engineering Reference
In-Depth Information
larly important if volume displacers are used, such as polydextrans [79], which
increase the effective target concentration but also the fluid viscosity [77].
Since at least 50% of the genes present in a genome are expected to be
expressed at less than 1 copy per cell and most of the others are present
in fewer than 10 copies, sensitivity will remain the key issue for this type
of analysis. With a push toward smaller sample sizes and ideally single cell
analysis, stochasticity in gene expression will become the ultimate limit [80],
requiring multiplexing of samples and arrays to overcome these statistical
hurdles. Finally, miniaturization and automation will provide some additional
solutions, as discussed in Chaps. 6 and 7.
11.4 Appendix
Assay Protocol for Expression Microarrays The following protocol is based
on methods worked out by the Biochemistry research group at Corning, Inc.
[50, 59, 75-77] and includes recent improvements.
1.
Reagents
•
5
×
FSS buffer: 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl
2
•
dNTP mix: 10 mM each of dGTP, dATP, and dTTP, 1 mM of dCTP
•
RevT solution: 8
µ
l5
×
FSS buffer, 4
µ
l 0.1 M DTT, 2
µ
ldNTPmix
and 1
µ
l of 1 mM dCTP-Cy3 or 1 mM dCTP-Cy5, and 2
µ
l of reverse
transcriptase
Universal Hybridization Kit (Cat. No. 40026, Corning Incorporated),
consisting in: Universal Wash Reagent A, Universal Wash Reagent B,
Universal Pre-Soak Solution, Sodium Borohydride Pre-Soak Tablets,
Universal Pre-Hybridization Solution, and Universal Hybridization
Buffer
Wash Soln 1: 50 ml Wash Reagent A, 447.5 ml water, 2.5 ml Wash
Reagent B
Wash Soln 2: 75 ml Wash Reagent A, 1425 ml water
Wash Soln 3: 300 ml Wash Soln 2, 1200 ml water
2.
Labelling of total human RNA
•
•
mix 1-5
g of random hexamers
(1 ug/ul) and nuclease free water; final volume 23
µ
l
µ
g of purified total human RNA, 3
µ
incubate for 5 minutes at 70
◦
C, quick chill on ice and spin down
•
l of RevT solution, mix well and incubate for 2 hours, 42
◦
C
•
add 17
µ
•
add 1
µ
l(2U/
µ
l) RNase H and 0.25
µ
l RNase A (30
µ
g/
µ
l); incubate
15 minutes, 37
◦
C
•
purify cDNAs with Qiagen's PCR purification kit and reduce the vol-
ume by evaporation to about 5-8
l
3.
Autofluorescence reduction and prewash:
•
µ
incubate slides in Universal Pre-Soak Solution with 1 tablet of NaBH
4
at 42
◦
C, 20-30 minutes, then transfer successively to Wash Solution 2
