Gene cloning and analysis by RT‐PCR.

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.

cDNA Synthesis Kits - SMART cDNA Synthesis - Clontech

cDNA Library Construction Kits - SMART cDNA Synthesis

Clontech's SMART cDNA Synthesis Kit - Biocompare

In an eukaryotic cell, the mRNA population constitutes approximately 1% of total RNA with the number of transcripts varying from several thousand to several tens of thousands. Normally, the high abundance transcripts (several thousand mRNA copies per cell) of as few as 5-10 genes account for 20% of the cellular mRNA. The intermediate abundance transcripts (several hundred copies per cell) of 500-2000 genes constitute about 40-60% of the cellular mRNA. The remaining 20-40% of mRNA is represented by rare transcripts (from one to several dozen mRNA copies per cell) [Alberts , 1994]. Such an enormous difference in abundance complicates large-scale transcriptome analysis, which results in recurrent sequencing of more abundant cDNAs.

A review of the Clontech's SMART cDNA Synthesis Kit

Transcriptional profiling using the common microarray platforms, whether in-house two colour slide based, Agilent, Affymetrix or Illumina all require microgram quantities of RNA and as such impose severe restrictionson the type of biological samples that can be used bystandard non-amplified protocols. However, with an ever-increasing need to utilise smaller andsmaller samples and address the relevant questions in the clinic, techniques have beendeveloped to amplify whole RNA populations from material such as clinicalbiopsy or laser capture microdisection and others. These techniques are now routinelyused by many commercial venders. Some of the more common approaches arereviewed here.

Clontech offers cDNA Library Construction Kits that utilize the SMART cDNA synthesis method to generate full-length cDNA.

cDNA synthesis/reverse transcription kit review - Labome

One of the greatest advantages of SMART technology is its increased efficiency compared to traditional technologies such as adaptor ligation. Its high efficiency and sensitivity enables you to use a very limited quantity of starting material, such as microdissected tissues, laser-captured cells, biopsy samples, etc. As little as 1-2 ng of total RNA is sufficient for generating a highly representative cDNA pool for different downstream applications.

SMART PCR cDNA SYNTHESIS Kit ..

With this experimental design, 130 plasmid clones were obtained. The digested cDNA clones were searched for sequence homologies in the GenBank DNA database by BLAST. DNA sequencing results of the digested 130 clones were found to have high homology to 14 genes known in the public database of the 14 kinds (). We identified 21 unknown genes, which were not homologous to any of the known genes in the Genbank database (). Among these 21 genes, we selected 7 genes which were over- expressed in glioblastoma, compared to non-tumor brain tissue (). To be considered over-expressed in glioblastomas, the genes tested had to show a threefold greater expression in tumor samples than in non-tumor brain tissue by semiquantitative RT-PCR. In this study, we considered these genes as candidates for over-expressed genes in glioblastoma.

Clontech offers cDNA Library Construction Kits that utilize the SMART cDNA synthesis ..

SMART cDNA Synthesis Kits by Takara Bio

SMART cDNA Synthesis Technology ensures uninterrupted cDNA synthesis, creating cDNAs with well-represented 5’ end sequences. Since terminal transferase activity and the subsequent SMART switching process occur preferentially at the 5' ends of eukaryotic mRNAs, truncated products resulting from premature termination of the reverse transcription reaction generally do not incorporate the SMART(er) oligonucleotide, and consequently are not amplified during PCR. Thus, cDNA pools created using our SMART technology and amplified by long-distance PCR are enriched for full-length cDNA. Additionally, because the 5’ SMART(er) sequence and modified oligo dT primer are not added onto genomic DNA or cDNA transcribed from ribosomal RNA, cDNA that is generated using SMART is free of these contaminating agents.

This technology is widely known as SMART™ cDNA synthesis (Clontech) ..

the 5' Full RACE Core Set cDNA Kit (Clontech, ..

- Rapid and reliable way to remove repeated transcripts from cDNA library
- Equalization of full-length-enriched cDNA before library cloning
- Simple procedure, no physical separation steps
- Fully compatible with Illumina / Solexa, ABI / SOLiD and Roche / 454 sequencing protocols

Read independent reviews on SMART cDNA Synthesis Kits from Takara Bio on SelectScience

SMART PCR cDNA Synthesis Kit User Manual - Gene X

To examine whether these new genes were expressed specifically in glioblastoma tissues, the expression patterns of transcription were examined by RT-PCR analysis for remaining 5 resected pairs of glioblastoma tissues and non-tumor brain tissues. The expressions of clone 25 were found to be relatively over-expressed as compared with non-tumor tissues (). The remaining six clones were not clear.