MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Provi...ng the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs.
Keywords:
Real time PCR / miRNA gene expression analysis / iron deficiency
TY - JOUR
AU - Timotijević, Gordana
AU - Milisavljević, Mira
AU - Nikolic, Dragana B.
AU - Milovanović, Bosko M.
AU - Nikolic, Dragana S.
AU - Nikolic, Miroslav
AU - Samardžić, Jelena T.
PY - 2015
UR - http://rimsi.imsi.bg.ac.rs/handle/123456789/848
AB - MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs.
PB - Društvo genetičara Srbije, Beograd
T2 - Genetika-Belgrade
T1 - Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis
EP - 416
IS - 2
SP - 405
VL - 47
DO - 10.2298/GENSR1502405T
ER -
@article{
author = "Timotijević, Gordana and Milisavljević, Mira and Nikolic, Dragana B. and Milovanović, Bosko M. and Nikolic, Dragana S. and Nikolic, Miroslav and Samardžić, Jelena T.",
year = "2015",
abstract = "MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs.",
publisher = "Društvo genetičara Srbije, Beograd",
journal = "Genetika-Belgrade",
title = "Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis",
pages = "416-405",
number = "2",
volume = "47",
doi = "10.2298/GENSR1502405T"
}
Timotijević, G., Milisavljević, M., Nikolic, D. B., Milovanović, B. M., Nikolic, D. S., Nikolic, M.,& Samardžić, J. T.. (2015). Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis. in Genetika-Belgrade
Društvo genetičara Srbije, Beograd., 47(2), 405-416.
https://doi.org/10.2298/GENSR1502405T
Timotijević G, Milisavljević M, Nikolic DB, Milovanović BM, Nikolic DS, Nikolic M, Samardžić JT. Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis. in Genetika-Belgrade. 2015;47(2):405-416.
doi:10.2298/GENSR1502405T .
Timotijević, Gordana, Milisavljević, Mira, Nikolic, Dragana B., Milovanović, Bosko M., Nikolic, Dragana S., Nikolic, Miroslav, Samardžić, Jelena T., "Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis" in Genetika-Belgrade, 47, no. 2 (2015):405-416,
https://doi.org/10.2298/GENSR1502405T . .
