Modification of LongSAGE for obtaining and cloning long concatemers.

BENCHMARKS The serial analysis of gene expression (SAGE) provides high-throughput information on researched samples. SAGE generates 9–13 bp tags from messenger RNA (mRNA) of the sample, and all tags are ligated to form concatemers for cloning and sequencing (1). LongSAGE uses similar procedures but a different restriction enzyme to obtain longer tags (19–21 bp). Both SAGE and LongSAGE tags are counted and statistically analyzed. The number of tags represents the expression of their corresponding genes. Large numbers of tags are required for more reliable statistical analysis of gene expression. The SAGE is being improved to increase the tag quality and quantity for analysis. For ditag PCR amplification, the primer pair was redesigned to prevent self-annealing and improve amplification efficiency (2). NlaIII digestion of PAGE-purified 102-bp ditags is an important step to produce 26-bp ditags for concatemerization. An extra purification step was added before NlaIII digestion to increase the efficiency of NlaIII digestion (3). LongSAGE tags (19–21 bp) were introduced to increase accuracy of tag identification (4–6). A heating step was added before gel electrophoresis to break contaminating aggregates (7). Low-cycle PCR amplification of the 3′ cDNA before BsmFI digestion and gel purification of the 3′cDNA after the BsmF1 digestion were introduced to generate high quantity and quality of tag/ditag (8). Robust-LongSAGE (RL-SAGE) was reported to increase the cloning efficiency by briefly digesting circular concatemers (9). Although these improvements enhance the SAGE efficiency, the number of long concatemers is still low. We report here two improvements to obtain cloned concatemers longer than 1 kb effectively: (i) briefly digesting the concatemers by NlaIII twice with an additional ligation between the digestions and (ii) selecting larger colonies. The procedures from RNA extraction to concatemer formation were performed by using the SAGE™ kit (Invitrogen, Carlsbad, CA, USA) or LongSAGE™ kit (Invitrogen). RNA extracted from different developmental stages of Lentinula edodes by TRI Reagent ® was the starting material for SAGE and LongSAGE. After NlaIII digestion, ditags were ligated to form concatemers and briefly digested using 1 μL NlaIII (10 U/μL; Invitrogen) in a total volume of 50 μL for 1 min at 37°C. The digested concatemers were self-ligated with the addition of 1.25 μL T4 DNA ligase (4 U/μL; Invitrogen) in a total volume of 10 μL at 16°C for 1.5 h and briefly digested using 1 μL NlaIII in a total volume of 50 μL again for 1–2 min at 37°C. The digested …