PubMed · 11239425
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K Sharma, C Y Peng. 2001. Spinal motor circuits: merging development and function.. https://doi.org/10.1016/s0896-6273(01)00208-2
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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.
In the previous experiment, we isolated and characterized glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of the oyster mushroom, Pleurotus sajor-caju. Expression levels of the GPD gene in the mycelia of P sajor-caju was significantly increased by exposing the mycelia to abiotic stresses, such as salt, cold, heat, and drought. We also showed that GPD confers abiotic stress resistance when introduced into yeast cells. The survival rate of the transgenic yeast cell that harbored the GPD gene was significantly higher when the yeast cells were subjected to salt, cold, heat, and drought stresses, compared with the yeast that was transformed with the pYES2 vector alone. In order to investigate the functional role of the P. sajor-caju GPD gene in higher plant cells, the complete P. sajor-caju GPD cDNA was fused into the CaMV35S promoter and then introduced into potato plants. Putative potato transformants were screened by using PCR. Twenty-one transformants were further analyzed with RT-PCR to confirm the expression of P. sajor-caju GPD. A RT-PCR Southern blot analysis revealed that 12 transgenics induced the P. sajor-caju GPD gene expression. A bioassay of these transformants revealed that the P. sajor-caju GPD gene was enough to confer salt stress resistance in the potato plant cell system. Results showed that P. sajor-caju GPD, which was continuously expressed in transgenic potato plants under normal growing conditions, resulted in improved tolerance against salt loading.
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