Hi,
I will work for quantfication of miRNA in plasma samples with real time TaqMan microRNA analysis. First of all, for extraction of miRNA, i choose (miRNeasy Serum/Plasma Kit For purification of total RNA, including miRNA, from animal and human plasma) and (serum miRNeasy Serum/Plasma Spike-In Control for normalization of miRNA purification from serum or plasma which is a 10 pmol lyophilized C. elegans miR-39 miRNA mimic: serves as exogenous control To allow for normalization of sample-to-sample variation in RNA isolation of plasma/or serum) provided by Qiagen. My question here, does i must choose the TaqMan microRNA assay for this cel-mir-39 as endogenous control for qRT PCR analysis, or i can choose others given by the database of thermofisher? if i must choose the assay of cel-mi-39 , does exist a particular caracteristics to select it because there are many choices!
Thanks
Hello Yosser
I advise you to analyze cel-mir-39 in your quatification Real-time PCR for decreasing variations between samples, beside this miR is from C.elegans and it does not have an overlapping expression with the rest of your miRs target for your study.
Best regards
i am sorry to ask a question regarding same issue. i am planing to use U6SnRNA as an endogenous control for my work on mouse system. am i going right?
or i have some better option for endogenous control in case of miRNA expression analysis by any method.
Hello Cash
I study the gene regulation by microRNAs in human monocyte and macrophage,as well as THP1 and HL60. For culture I advise you try with RNU48, U6 and RNU44. But for experiments in plasma, you should use a Spike-In Control as cel-mir-39.
Please, check this file
Best regards
hi oscar- thanks for the reply. but i need to use this endogenous control in case of mouse tissue and i dont find u6sno in the given datasheet.
and one more thing i like to ask to both of you (oscar and yosser)- please tell me about the effect of primer concentration used for cDNA synthesis and real time PCR because i think this is the one that could be critical in getting true expression values.
Hello Cash;
I think that concentration of primers or assays taht you will used in your analysis, it all depends on the protocol provided by any kit you used for the quantification ( for example qiagen, miVana , taqman.. ) or any kit ! you must follow it.
hi yosser,
nice to get your response, unfortunately i am not using any kit for this purpose. trying as manual as possible. as i have seen the fluctuation in getting differential expression of miRNA quantification based on using different concentrations of primer used for RT reaction.
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