The lncRNA qPCR service covers lncRNA and mRNA. In addition to the general qPCR design guidelines, special design considerations are given to lncRNA. The qPCR primers are situated as closely as possible to the array probe location chosen to target the unique exon or splice junction site of the transcript (Fig. 1). If the LncRNA is a natural antisense LncRNA, a strand specific reverse transcription primer, instead of oligo(dT) primer, is used for the first strand cDNA synthesis. Preferably, the RNA prep is the same as used for the microarray profiling experiment.
When selecting differentially expressed lncRNAs for qPCR confirmation, the magnitude of fold change, p-value (adjusted), and raw signal intensity are evaluated. Genes showing a significant FC and p-value but having a very low raw intensity may still not be confirmed by qPCR. Empirically, at least one of the group raw intensities is greater than 200~500.
Figure 1. Many LncRNAs have multiple isoforms, with different expression patterns and functions, or overlap with neighboring protein-coding genes. To solve this problem, Arraystar designs transcript-specific probes in the LncRNA expression array. To correctly validate array data, PCR primers should also be designed for detecting specific transcripts as shown in Design B, which distinguishes LncRNA-X from another isoform of LncRNA-X.