PubMed · 8650369
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N Fujimoto, S Kawamoto, K Okubo. 1996. [Gene expression analysis by random cDNA sequencing].. https://pubmed.ncbi.nlm.nih.gov/8650369/
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STALARD (selective target amplification for low-abundance RNA detection) enables isoform-level quantification of low-abundance RNAs using conventional laboratory equipment. Here, we describe steps for RNA isolation, primer design, reverse transcription, selective target amplification, and downstream analysis. The protocol couples selective pre-amplification with a quantitative reverse-transcription PCR (RT-qPCR) readout and optional nanopore sequencing. Using 1 μg input RNA and 12 pre-amplification cycles, STALARD reduces Cq values by approximately 10-12 cycles, bringing the target into a reliably quantifiable range. For complete details on the use and execution of this protocol, please refer to Jeong et al.1.
The mac25 cDNA was originally cloned from leptomeningial cells and subsequently reisolated through differential display as a sequence preferentially expressed in senescent human mammary epithelial cells. The deduced amino acid sequence of the human mac25 propeptide shares a 20-25% identity to human insulin-like growth factor-binding proteins (IGFBPs), suggesting that mac25 could be another member of the IGFBP family. In the present study, we have generated recombinant human mac25 (rh-mac25) in a baculovirus expression system and assessed its affinity for IGFs and have evaluated the pattern of expression of the mac25 gene in human tissues. Binding of 125I-IGF-I and 125I-IGF-II to rh-mac25 was demonstrated by Western ligand blotting after nondenaturing polyacrylamide gel electrophoresis and by affinity cross-linking with as little as 2 nM rh-mac25. Specificity of rh-mac25 binding to 125I-IGFs was demonstrated by competition for rh-mac25 binding with unlabeled IGFs, but not with [QAYLL]IGF-II analog, which has 100-fold less affinity for IGFBPs. In comparison with IGFBP-3, rh-mac25 has at least a 5-6-fold lower affinity for IGF-I and 20-25-fold lower affinity for IGF-II. mac25 mRNA was detectable in a wide range of normal human tissues, with decreased expression in breast, prostate, colon, and lung cancer cell lines. In conclusion, mac25 specifically binds IGFs and constitutes a new member of the IGFBP family, IGFBP-7. Its wider distribution in normal tissue and lower expression in several cancer cells indicate that IGFBP-7 may function as a growth-suppressing factor, as well as an IGF-binding protein.
We previously reported that Nicotiana benthamiana plants transformed with the wild-type 54-kDa region of the pepper mild mottle tobamovirus, S strain (PMMoV-S), displayed two different resistance responses against PMMoV infection. Some of the transgenic plants exhibited a complete and highly resistant phenotype while the remaining plants showed a delayed resistance (Tenllado et al., 1995, Virology 211, 170--183). Here we show that some of the N. benthamiana plants transformed with a construct expressing a PMMoV-S truncated 54-kDa protein coding sequence also displayed a complete and highly resistant phenotype similar to that shown by the wild-type 54-kDa transgenic plants. This result indicates that the wild-type, full-length 54-kDa protein is not required in mediating the complete resistance phenotype against PMMoV. The remaining truncated 54-kDa transgenic plants were susceptible to PMMoV infection but showed a variable delay in the appearance of symptoms. Unlike the wild-type 54-kDa transgenic plants, which were initially susceptible to the infection but recovered later, the truncated 54-kDa transgenic plants never exhibited this delayed resistance phenotype. However, they displayed a new type of altered symptomatic phenotype. The truncated 54-kDa transgenic lines also exhibited a lower level of transgenic transcripts compared to the wild-type 54-kDa transgenic lines which could account for the absence of the delayed resistance phenotype.