Normalized and Subtracted cDNA Libraries
Normalized libraries
.
One way to avoid the repetitive and expensive
sequencing of highly redundant cDNAs is to use a normalized library.
Normalization equalizes the representation of all transcripts in the cDNA
population (overview
of the method). Thus the identification of novel and rare transcripts is
enhanced (Table 1). Additionally,
cDNA from different tissue sources can be independently tagged during cDNA
synthesis for identification of transcript source after
sequencing.
Subtracted libraries
During mass sequencing of normalized cDNA
libraries in the Keck Center, redundancy is continuously
monitored by sequence assembly. When identification of novel transcripts falls
below 50%, a molecular subtraction of the already sequenced clones is
performed (overview
of the method). The resulting subtracted library is enriched for low abundance transcripts that are commonly
missed in conventional sequencing (Table 1). The technique is also used to
subtract commonly expressed clones from previous
libraries.
Table 1. Data from normalized and subtracted
libraries constructed in the Keck Center.
|
|
Normalized |
|
Subtracted |
Library |
# Sequences |
% Unique cDNAs |
|
Total # of
Sequences |
% Unique cDNAs |
|
Zebra Finch (brain) |
18,240 |
59% |
|
35,000 |
49% |
|
Planarian |
5,000 |
60% |
|
16,320 |
57% |
|
Maize
(root) |
6,000 |
65% |
|
|
|
|
Horse
(cartilage) |
16,319 |
59% |
|
|
|
|
Corn
Rootworm |
11,149 |
48% |
|
14,921 |
50% |
|
Pig
(uterus, hypothalamus) |
10,000 |
60% |
|
|
|
|
Cattle
(placenta) |
5,047 |
68% |
|
12,777 |
64% |
|
Honey Bee
(brain) |
7,968 |
60% |
|
15,311 |
59% |
|
Cassava |
8,956 |
66% |
|
|
|
|
Cassava
drought |
9,210 |
60% |
|
|
|
|
Leafy
Spurge |
10,226 |
44% |
|
20,928 |
55.4% |
Number of sequenced
clones and percentage of cDNAs identified as unique upon assembly in normalized
libraries and, in some cases, after subtraction and further sequencing. In both
normalized and subtracted libraries the identification of unique sequences is
substantially enhanced.
Materials provided by the
researcher:
-
Tissue (2-10g
depending on the source), or
-
High-quality
total RNA (1-2mg)
Services performed by the Keck
Center:
-
Isolation of total RNA and poly(A) RNA
-
cDNA synthesis
and size fractionation from 0.5kb (unless otherwise indicated by the
PI)
- Cloning into
pBluescript II or your vector of choice
-
Transformation
into E.coli electrocompetent cells
-
Normalization
-
Sequencing of
192 clones. Vector and quality trimmed sequences are assembled to determine
sequence similarity and redundancy and identity is determined by comparison to
specific databases.
Subtraction is normally performed for libraries constructed and
sequenced in the Keck Center. The efficiency of subtraction is determined by
sequencing 192 clones as described above.
Custom services will be considered
Pricing
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