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Get Longer cDNA (and aRNA) with this Reverse Transcriptase:
ArrayScript™ M-MLV Reverse Transcriptase
By Dana Broussard and Bret Schneider, Production Division,
Ambion, Inc.
ArrayScript™ (patent pending) is an M-MLV reverse transcriptase (RT) specifically engineered to produce high yields of full-length cDNA. Up to twice as much amplified RNA (aRNA) is synthesized in RNA amplification experiments from low amounts of input total RNA (~100 ng) when first-strand cDNA is generated with ArrayScript compared to four other RTs [1, 2]. In addition to RNA amplification and gene array analysis, ArrayScript can enhance a variety of other molecular biology techniques, including RT-PCR and RACE procedures. Here, we report on the greater length of cDNA products (Ambion's FirstChoice® RACE- and PCR-Ready cDNAs) that result from reverse transcription of human and mouse tissue poly(A) using ArrayScript.
ArrayScript™ vs. Wild Type M-MLV RT
The FirstChoice® RACE- and PCR-ready cDNA products are a mixture of cDNA populations separately primed with both random decamers and oligo(dT) to maximize full-length cDNA. Both products begin with the same high quality FirstChoice Poly(A) RNA and are purified identically; the RACE-Ready cDNAs undergo additional pre-cDNA synthesis processing to maximize representation of 5'- and 3'-end sequences. In the experiments described here, FirstChoice Poly(A) RNA was reverse transcribed using either ArrayScript or wild type M-MLV RT, purified to remove primers and small reaction products, and assessed on an Agilent® 2100 bioanalyzer.
Get Longer cDNA Regardless of Primer
All RACE- and PCR-Ready cDNA preparations
synthesized with ArrayScript had a longer average cDNA length
when compared to samples reverse transcribed with wild type M-MLV
RT (Figure 1). Average length varied with species, tissue, and
primer used. For example, average cDNA lengths were 31-93% longer
when primed with oligo(dT) (Figure 1, Panel A) and 7-15% longer
when primed with random decamers (Figure 1, Panel B). While these
were single data points, the data represents 8 distinct samples
that all show increase in length. As expected, the increase in
average cDNA length is smaller for random primed products, because
primers that anneal close to the termini can only produce finite-sized
cDNAs.
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Figure
1. Longer
Average cDNA Length after Reverse Transcription Using
ArrayScript™. cDNAs
were prepared from 2-7 µg (after adapter
ligation; RACE-Ready) or 5 µg (PCR-Ready) of
FirstChoice® Poly(A) RNA. The RNA
was reverse transcribed using either wild type M-MLV
(200 U/µl) or ArrayScript (200 U/µl), and
oligo(dT) primers (A) or random decamers (B).
After column purification to remove primers and small
reaction products, cDNA length was assessed using
an Agilent® 2100 bioanalyzer. Results
are expressed as average cDNA length for a given
sample. |
Longer cDNA Gives You the Advantage
The increased length of the cDNA population should be especially beneficial to researchers using RACE procedures to identify either the 5' or 3' ends of messages and for researchers constructing cDNA libraries. Identification of genetic isoforms and splice variants should also be enhanced by using a longer cDNA population. Thus, by producing significantly longer cDNAs, ArrayScript not only is the enzyme of choice for RNA amplification and gene array analysis, but also enables synthesis of longer products that can benefit a variety of molecular biology techniques.
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