Hi Minseon, the gene expression of a given mature miR can be measured by qPCR through estimation of its quantity in the cytoplasm. As the transfection of the miRNA inhibitor will result in the act of this inhibitor over the mature miRNA, when the sample that was transfected with the miRNA will be quantified to that mature miRNA is expected that the gene expression for that miRNA would be decreased, exactly because the action of the inhibitor. To guarantee that this is the case, you can try to quantify the pre-miRNA and the pri-miRNA that gave origin to that inhibited mature miRNA, the levels of the pri and pre-miRNA for that mature miRNA will not change once the inhibition was done just in the level of mature miRNA. The case of the negative control it not occur the same level of gene expression than seen in the transfected inhibited one due it was used only the lipofectamine, no inhibitor was present, so basically the gene expression of the miRNA will be the same of the control.

At least this was the case I found in my experiments that I done during my doctorate.

I hope that it helps you.

Pedro

One bias that I think could be linked to the estimation of the gene expression of the mature miRNA is the use of the U6 snRNA as the housekeeping gene. This because, the U6 is found as the result of the process of splicing inside the nucleus, while the mature miRNA are formed in the cytoplasm, so both suffered actions of different enzymes, besides the question of the nuclei barrier between them, and furthermore don’t have the same function while are non-coding RNAs.

Pedro Paulo Gattai Thank you for your kind explanation. I think that it might be affected by the experimental conditions under which the RT-qPCR is performed can influence the outcome. For example, some protocols include steps to denature double-stranded RNA complexes, potentially allowing detection of the miRNA.

RT-qPCR typically detects single-stranded RNA, as the reverse transcription step converts RNA into complementary DNA (cDNA). If the miRNA is bound to an inhibitor and forms a double-stranded complex, it might not be efficiently reverse-transcribed into cDNA.

I think I need to check my conditions.

Cheers,

Minseon

Yes Minseon Park , indeed the experimental conditions can influence the outcome undoubtly. When I did my first try to extract the miRNAs from my tissue samples during my doctorate, I did the protocol for miRNAs extraction of the commercial kit from Ambion with the filter columns until the last step described in its manual, but at that moment I didn’t recovery the miRNAs, and it was clear due to the qPCR results after the cDNA preparation. The results showed that the miRNAs, both rno-miR-26a-5p and rno-U6-snRNA, didn’t present any significant value. Then, I questioned this with the advisory department from the manufacturer. And they told me that during the Ambion kit extraction, I shouldn’t do the entirely protocol, instead I should stop some steps backwards, and this because if I did the protocol until its end, in one of the last steps I would washing out the short RNAs and consequently would discard them. So, after that all my extractions I did as they suggested and could recovery the total RNA, including the small ones.

Citing their words:

”The initial purification steps with the kit are the same if will be done the enrichment for small RNAs or not. First, the sample lysis then, in sequence, does the extraction with phenol: chloroform, which already excludes the DNA. The following steps differentiate themselves if will be made the enrichment or not, but in both procedures the sample will be purified in the column. The difference is that, when didn’t perform the enrichment, after the ethanol addition in the sample (with an end concentration of around 55%), the sample passes only one time in the column, which will immobilize the total RNA, after the washes are eluted. In the case of opting by enrichment, in the first column filtering, the larger RNAs will be immobilized and the small ones will be collected in the filtering, basically why the ethanol final concentration will be low, of 25%, which favors the immobilization in the column of the greater and the passage of the small ones. In this filtered, then, increase the ethanol concentration (end concentration of 55%), before adding the sample in the second column, in which there will immobilization of sample RNAs, which already have the majority of small molecules. The small RNAs, then, after the washes, will be eluted. 

In this way, the enrichment of small RNAs is made by selective filtering resulting from diverse concentrations of ethanol in the sample. Furthermore, the columns from the kit have the property to retain small RNAs, once, generally, for common kits for RNA purification, which don’t are specific for small RNAs, the columns don’t have the capacity to retain molecules with minus than 100 bases. The columns from this kit retain molecules from 10 Mers until 1 kb.”