What is the concentration of mRNA and miRNAs that is possible to recover from saliva? Are miRNAs degraded (or not) after 2-3 days if properly stabilized (i.e. RNALater or similar)?
MicroRNAs (miRNAs) are small RNA molecules that regulate post-transcriptional gene expression by binding messenger RNAs (mRNAs), blocking their role in translation or marking them for degradation. To date, computational methods for predicting mRNA targets have assumed an all-or-nothing mode of miRNA-mRNA interaction. Here we introduce a computational approach that predicts the degree of interaction, taking into account initial miRNA and mRNA concentrations. Using this approach, we can predict whether specified interactions are likely to be functionally relevant within physiologically relevant concentration ranges.
Research data clearly show two distinguishable factors that influence usefulness of RNA isolated from FFPE sections. Under the assumption that rRNA and other RNA species (most notably mRNA) show similar degradation kinetics, integrity of RNA can be assessed using electrophoresis methods, and depends mainly on the quality of the fixed tissue sample (time between resection and effective fixation), storage time and conditions of the paraffin blocks (Fig. 1), and presumably embedding conditions (primarily the incubation time and temperature in hot paraffin). The second factor, chemical modification of RNA by formaldehyde, is not resolved by electrophoresis assays, but has a strong negative effect on RT-PCR, and presumably other enzymatic procedures as well. Because the actual PCR template is newly synthesized cDNA, the observed negative effects in our assays must be due to inhibition of the reverse transcription reaction. The degree to which chemical modification impacts RNA quality depends on fixation time (Fig. 3), and also on the RNA isolation procedure.
The RNA isolation procedure we used involves a short proteinase K digestion, followed by incubation at 70°C under conditions that promote breakage of methylene crosslinks which result from formalin fixation. Proteinase K digestion is necessary to release RNA from the meshwork of crosslinked protein and nucleic acids by digesting the protein portion of the crosslinked molecules, potentially down to the level of tetrapeptides [12]. However, it does not attack the actual methylene bridge that forms the crosslink, because it does not involve a peptide bond. In contrast, the heating step breaks the actual crosslinks, but is limited by the heat tolerance of the RNA itself, and by the irreversible nature of part of the crosslinks [2]. Accordingly, it can be assumed that the inhibitory effect of RNA from FFPE samples on cDNA synthesis that we observed is due mainly to very short peptides that are irreversibly crosslinked to RNA bases.
Even though integrity of RNA isolated from FFPE specimens clearly deteriorates with storage time, performance in RT-PCR assays is relatively poor even for seemingly intact RNA, and changes relatively little during the first 12 months of storage at room temperature–indicating that the limiting factor is chemical modification by formaldehyde.
depending types of mRNA their stability vary in 20 sec to hours. but miRNa stability is unknown until now.
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- Biological Science
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- Nucleic Acids
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