Identification of Rare Alternative Splice Variants with Full Spectrum™
Transcriptome-Wide RNA Amplification
In genomic research today, there is
an increased interest in identifying and quantifying of alternatively
spliced transcripts. Now it appears that as many as 75% of the estimated
30,000 gene transcripts are alternatively spliced. Alternative splicing has
been found to be important for various normal cellular activations such as
development, differentiation, and programmed cell death. Alternative
splicing has also been found associated with disease phenotypes such as
hypertension, cancer, and even obesity. Identification and analysis of
alternative splicing can be difficult for several reasons, among which are
the following
Alternative splicing can occur all
along a gene transcript. Both 5’ and 3’ ends of the mRNA sequence need to be
preserved. This can be a problem if the gene transcript is very long or if
secondary structure impedes reverse transcription.
Some alternative splice variants are
very low in abundance in many cells. Studies of low abundance transcripts
require an amplification step, as has been done in the following UCLA study.
Experimental Determination of Splice Variants
A recent study was undertaken by UCLA scientists to experimentally verify
splice variants predicted from EST database analysis to be tissue-specific
and mouse strain-specific. An example of one such gene’s transcripts is
presented below.
Fig 1.
Genomic representation of splicing patterns for brain/testis-specific and
mouse strain-specific transcripts (at left), and predicted transcript sizes
by EST database analysis (at right).
The starting amount of total RNA for the
unamplified RT reaction was 1 µg.
First strand cDNA was primed with both oligo-dT and random hexamers. To
amplify RNA, 0.5 µg
was used in our Full Spectrum™ RNA Amplification kit protocol with 15 cycles
of amplification. PCR products generated using forward and reverse primers
(Fig. 1) were sequenced to verify alternative splicing.
With Full Spectrum™ RNA Amplification technology, you can:
Identify and quantify alternative splicing in both long and rare gene
transcripts
Examine differential gene expression in clinical samples in which the RNA
may be partially degraded
Examine gene expression in small numbers of cells such as those obtained
from laser capture microdissection.
Amplify RNA with no purification steps, using fewer tubes, and with fewer
pipetting steps using Full Spectrum’s 1-tube, 2-step, 3-hour protocol
Our Full Spectrum™
line of RNA Amplification products can help facilitate alternative splicing
research:
