Applications such as northern blot analysis, cDNA library construction and cDNA subtraction are demanding when it comes to starting material. For these techniques to be successful, it can often require as much as 5 ug of poly A+ RNA. A solution to this problem can be found in Clontech’s SMART cDNA Synthesis Kit. This technology is an extremely useful tool for anyone who wants to perform techniques that require cDNA synthesis but where starting material is limited. SMART cDNA synthesis technology allows the production of double-stranded cDNA (dscDNA) to be performed with as little as 50 ng of total RNA.
SMART cDNA synthesis takes advantage of the template switching ability of RNAse H- point mutants of MMLV Reverse Transcriptase (such as Clontech’s Powerscript or LifeTech’s Superscript). Poly A+ RNA is reverse transcribed from a modified oligo dT primer. When the RT reaches the 5’ end of the RNA, its terminal transferase activity adds a short deoxycytidine (dC) sequence. This dC sequence then anneals to the 3’ end of the SMART oligonucleotide (containing a stretch of dG), at which time the RT jumps to transcribe the 5’ portion of the SMART oligonucleotide. The result is single stranded cDNA flanked by the modified oligo dT primer on the 3’ end and the SMART oligonucleotide on the 5’ end. Long distance PCR is then performed on the single-stranded cDNA using primers complementary to regions in the oligo dT sequence and the SMART oligonucleotide. Because the 5’ priming site for PCR is 5’ to the start of the cDNA, full-length products are effectively enriched.
During the process of generating dscDNA, a careful analysis of the PCR optimization is necessary to avoid over-amplification. Over-amplified cDNA tends to manifest as a shift toward unusually high molecular weight products. Once the process has been optimized, the resulting dscDNA can be used for a number of different applications, including cDNA library construction, cDNA subtraction and "Virtual" Northern Blot Analysis.
In my hands, this technique has worked extremely well. Although I have not tested the lower limits of starting material, I have been able to get consistently good results using 500-1000 ng of total RNA from freshly isolated T-lymphocytes (which is a typical yield from approximately 1 million cells). This SMART amplified cDNA was used for both cDNA subtractions and for "Virtual" Northern blot analysis and for both techniques I was happy with the results that were obtained.
That’s not to say that the SMART cDNA Synthesis Kit is perfect. When using this kit, beware of the included Chromaspin 1000 columns for purification of your cDNA. Unless size exclusion is an absolute necessity, much better yields can be achieved with a standard PCR purification method (such as Clontech’s Nucleospin kit or Qiagen’s Quiaquick PCR purification system). In addition, as is usually the case with new technologies, the kit runs on the expensive side. Each kit contains enough reagents for 7 reactions and goes for $465. Despite this, when there is a limited amount of starting material and there are interesting experiments to be done, the SMART kit is the way to go.