Biomedical Engineering Reference
In-Depth Information
Figure 5.5
Single-cell profiling by Global-RT-PCR. Three individual cells from each of two groups
were obtained and the RNA was amplified and profiled on Agilent 44k Whole Human Genome
arrays. After a
t
-test was performed to identify a list of 358 genes which distinguished between
the two groups, the gene panel was subjected to hierarchical clustering. Reproducible results
from each of the cells used in each of the groups were obtained and provided a strong identifier
panel. (See Plate 5.5.)
the first round of global-RT-PCR, providing an excellent resource for validation either by
downstream quantitative PCR or use on another array type. A final advantage is that because
the method is 'home-brewed' it is cost effective. The reaction components are inexpensive
and the entire reaction can be run for under $50. One of the key developments that make
the global-RT-PCR procedure possible is the ability to control the length of the cDNAs
generated during the RT reaction. This is accomplished by running a very short (5min) RT
reaction compared with the 2 h used in the T7 amplification [31, 32, 34]. By keeping this
step short, the average length of the products that result fall within a narrow range, on the
order of only 300 bp in length. This ensures that, during the PCR step, each template is
processed with similar efficiency, leading to improved reproducibility and preserving the
abundance relationships of the original RNA pool.
PROTOCOL 5.8 Reverse Transcription
Equipment and reagents
Lysis buffer (52 m
M
Tris-HCl,pH8.3,78m
M
KCl, 3.1m
M
MgCl
2
, 0.52% (v/v) NP-40)
•
SUPERase-In (Ambion)
•
