The In-Fusion SMARTer Library Construction protocol can be completed in fewer steps than other library construction methods due to a highly efficient cDNA synthesis and cloning process. Only three enzymes are required to complete the entire protocol, as opposed to the usual 6–8 enzymes required for other methods. The SMART(er) cDNA synthesis protocol is user friendly and straightforward with no adaptor ligation or tailing steps. Your precious RNA is subjected to the to the least possible handling, thereby minimizing the risk of degradation.
In-Fusion Cloning makes it easy to clone your SMARTer cDNA library into the pSMART2IF or pSMART2IFD linearized vectors (included in In-Fusion SMARTer kits) in just one 30 min reaction. Most importantly, since In-Fusion Cloning is designed to join fragments of DNA with 15 complementary bp at their ends, In-Fusion kits can be used to precisely transfer your SMARTer cDNA into ANY linearized vector. If you would like to clone your library into your own expression vector for functional analysis, simply amplify your vector by inverse PCR using primers that create linear vector ends that are complementary to the ends of the SMARTer cDNA. Primers must have two characteristics: the 5’ end of the primer must contain 15 bases that are complementary to 15 bases at one end of the DNA fragment to which the vector will be joined (i.e., the insert), and the 3’ end of the primer must contain sequence that is specific to the target vector.
During first-strand SMARTer cDNA synthesis known universal primer sequences are incorporated at both ends of the cDNA. Following first-strand synthesis, SMARTer cDNA is immediately available for PCR amplification.
The SMART cDNA Library Construction Kit is designed for the cloning of full-length cDNA into a phage TriplEx2 vector. The kit combines the SMART technology (Switching Mechanism At 5' end of RNA Template) for cDNA amplification with adaptor-free, directional cloning into the TriplEx2 vector. This kit contains two separate protocols, allowing you to choose a method based on your starting material. The first protocol employs long-distance PCR (LD PCR), for researchers limited by their starting material. As little as 50 ng of total RNA can be used as starting material (1). The second protocol provides a more straightforward protocol for researchers with abundant amounts of starting material (i.e., 1 µg or more of poly A+ RNA). SMART libraries contain a higher percentage of full-length clones than libraries constructed by conventional methods or other full-length cDNA synthesis protocols. Thus, clones isolated from SMART cDNA libraries contain sequences corresponding to the complete 5' untranslated region of the mRNA (2).
SMART (Switching Mechanism at 5’ End of RNA Template) is a unique technology that allows the efficient incorporation of known sequences at both ends of cDNA during first strand synthesis, without adaptor ligation. The presence of these known sequences is crucial for a number of downstream applications including amplification, RACE, and library construction. While a wide variety of technologies can be employed to take advantage of these known sequences, the simplicity and efficiency of the single-step SMART process permits unparalleled sensitivity and ensures that full-length cDNA is generated and amplified.
Neither coefficient reflects the true significance of the discrepant values. From a technical point of view, there are a number of steps in the procedure that can be the source of these discrepancies. From a biological perspective, the complexity of the mRNA template and the efficiency of the enzymatic reactions may also be the explanation for these discrepant values. There have been attempts to preserve mRNA complexity by increasing the efficiency of the RT reaction. Amplification can also be a source of variability in the experimental procedure. All three probe labeling approaches we used here involve an RT step, and all three probes had similar levels of discrepant gene detection. This argues that the discrepant values are due to sample complexity and RT, rather than amplification. The Smart PCR cDNA method was originally developed to produce a high-quality, full-length cDNA for library construction. Recently, the Smart PCR cDNA synthesis was used to confirm differentially expressed genes identified on microarrays.
Synthesis of labeled cDNA probes from preparations of total RNA is the most promising alternative to purified mRNA for array applications. Indeed, CLONTECH Laboratories (Palo Alto, CA) and Research Genetics (Huntsville, AL), both manufacturers of filter arrays, include protocols in the user manuals for the preparation of cDNA probes from 0.5–10 μg of total RNA. Polymerase chain reaction (PCR)-based cDNA methods for amplification from limited amounts of RNA are also being used for differential gene expression profiling. The SMART PCR cDNA synthesis method (CLONTECH Laboratories) was used in gene expression profiling experiments to produce cDNA libraries from total RNA that were representative of the mRNA. In vitro transcription of heterogeneous cDNA synthesized from total RNA has been modified to incorporate biotin into the probe and adapted to high-density arrays. Key to these alternative approaches is a reproducible and representative synthesis of complex cDNA probes derived from the mRNA population present in the original sample.
Digoxigenin-labeled cDNA probe from total RNA was synthesized as described above, with the following modifications. Five micrograms of total RNA was reverse transcribed with oligo d(T) for 2 hours at 42°C, with an additional 200 U Superscript II added after the first hour. The labeled cDNA was treated with 2 U each RNase H and RNase A (Roche Molecular Biochemicals) for 20 minutes at 37°C. One microliter of the 20-μl RT reaction was evaluated for dig-11-dUTP incorporation by denaturing acrylamide electrophoresis. The remaining 19 μl of the preparation was used for hybridization. This type of digoxigenin-labeled probe will be referred to as the total RNA probe in subsequent sections.