Final answer:
The observed down-regulation of a gene in total RNA but not in mRNA in qPCR experiments could be due to the presence of unprocessed or degraded RNA in the total RNA sample and the selective enrichment of mature mRNA during the purification process.
Step-by-step explanation:
Your qPCR results showing a gene down-regulated in the total RNA but not in the mRNA may be due to several reasons related to RNA purification and post-transcriptional modifications. RNA is inherently very unstable and prone to degradation by RNases, which are ubiquitous in nature. The extraction of RNA involves using buffers and enzymes to inactivate other macromolecules and protect the RNA. However, not all RNA extracted will be mRNA; total RNA includes rRNAs, tRNAs, and other non-coding RNAs.
During the mRNA purification from total RNA, techniques such as column purification are often employed. These can separate mRNA from other forms of RNA based on differences in size and charge. However, it's important to note that the total RNA sample may contain RNA molecules that are not fully processed or are degradation products, which could contribute to different gene expression levels when compared to purified mRNA. The mRNA purification step may selectively enrich for fully processed, mature mRNA transcripts ready for translation, which might not show the same level of down-regulation as observed in the total RNA sample.
Post-transcriptional modifications, such as RNA splicing, remove introns and can regulate gene expression by dictating which RNA transcripts are available for translation into proteins. The gene in question might be present in the total RNA sample as unprocessed or partially processed precursors that are ultimately degraded and therefore appear as down-regulated, whereas in the mRNA sample, only mature transcripts are measured, and these might not be down-regulated.