Roles of human milk miRNAs and microbiota in infants gut: a systematic review

Early-life breastfeeding practice provides multiple advantages to infant subsequently contributing to significant short- as well as long-term positive health outcomes. The current notion suggested that in the first 1000 days of life, infant’s body is susceptible to external factors and highly inducible towards complex repertoire of components originated from mother’s milk. This condition permits diverse components of human breast milk ranging from microRNA and microbiome to larger size macronutrient to act by supplementing and supporting their under-developed body system in early life. Currently, the discoveries of milk miRNA as key effectors in multiple biological processes in different infant’s organs including gastrointestinal tract are growing rapidly. In addition, the perception on the translocation of bacteria from mother’s intestine to breast milk has given rise to the idea that these bacteria may as well dynamically transferred to the infant and provide protection to poor barrier integrity of their gut. Hence, the aim of this paper is to conduct a systematic review in order to collect, compile, and extract data related to human milk miRNAs and microbiota as well as their functions in infant’s gut following the relevant guidelines of Preferred Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA). A systematic search will be done on at least three databases such as Scopus, PubMed and Medline using relevant Medical Subject Headings (MESH) terms including human milk AND miRNA AND (microbiota OR microbiome) AND (infant OR child OR children) AND (gut OR intestine OR intestinal) from 2011 through 2021. From the databases, papers obtained will be sorted accordingly based on the objective, and inclusion and exclusion criteria and subject to Crowe Critical Appraisal Tool (CCAT) to further assess and ensure the quality, validity and eligibility of the research papers before reviewing thoroughly. This study is expected to provide a comprehensive presentation and information on the types of miRNAs and microbial species as well as their roles, functions and importance in boosting the system within infant’s gut. In depth, the miRNAs that capable of inducing genetic expression of their target genes or regulate particular important signalling pathways that would be beneficial in enhancing the immune system within infant’s gut may be presented in this study. Similarly, for some beneficial microbial species in the gut such as Lactobacilli with unique mechanisms and mode of actions in preventing the colonization of pathogenic bacterial within infant’s gastrointestinal tract and reduce clinical infection

Specific microRNAs among milk siblings: an epigenetics approach towards understanding the basis of milk kinship

Milk kinship is an Islamic belief described as a relationship established when infants receive breast milk from non-biological mothers. This form of kinship is said to bear a very close resemblance to blood relation whereby the recipients’ infants are regarded as milk siblings to the biological children of the breastfeeding mother. Any future marriage between these individuals is forbidden likewise between the recipient infant and the nursing mother herself as they are thought to have a form of consanguinity. The consanguinity formed by virtue of milk sharing might be due to the composition of human breast milk, especially milk microRNAs that are responsible for the epigenetic modulation of gene expression. miRNAs can regulate gene expression by modulating genome-wide epigenetic status of genes, and similarly-shared genes might be the basis that has led to milk kinship formation. Thus, the objective of the present study is to identify potential lactationspecific miRNAs that are similarly shared among milk siblings and their nursing mothers. The study began with molecular extraction of milk RNA from the nursing mothers and cell-free plasma RNA from all milk siblings and their nursing mothers. The RNAs extracted from both sample types were further analyzed using NanoString nCounter® miRNA Panel Analysis (NanoString Technologies, Seattle, WA) to measure the abundance of individual miRNAs biomarkers present within the samples. This study is expected to provide scientific explanation that could divulge the secrets behind milk kinship establishment with thorough presentation on the lactation-specific miRNAs shared between milk siblings. Hence, the way for future research would be paved, making the development of milk kinship identification tool possible

Specific MicroRNAs among milk siblings: an epigenetics approach towards understanding the basis of milk kinship

Introduction: Milk kinship is an Islamic belief described as a relationship established when infants receive breast milk from non-biological mothers. This form of kinship is said to bear a very close resemblance to blood relation whereby the recipients’ infants are regarded as milk siblings to the biological children of the breastfeeding mother. Any future marriage between these individuals is forbidden likewise between the recipient infant and the nursing mother herself as they are thought to have a form of consanguinity. The consanguinity formed by virtue of milk sharing might be due to the composition of human breast milk, especially milk microRNAs that are responsible for the epigenetic modulation of gene expression. miRNAs can regulate gene expression by modulating genome-wide epigenetic status of genes, and similarly-shared genes might be the basis that has led to milk kinship formation. Thus, the objective of the present study is to identify potential lactation-specific miRNAs that are similarly shared among milk siblings and their nursing mothers. Methods: The study began with molecular extraction of milk RNA from the nursing mothers and cell-free plasma RNA from all milk siblings and their nursing mothers. The RNAs extracted from both sample types were further analyzed using NanoString nCounter® miRNA Panel Analysis (NanoString Technologies, Seattle, WA) to measure the abundance of individual miRNAs biomarkers present within the samples. Expected Outcomes: This study is expected to provide scientific explanation that could divulge the secrets behind milk kinship establishment with thorough presentation on the lactation-specific miRNAs shared between milk siblings. Hence, the way for future research would be paved, making the development of milk kinship identification tool possible

Effects of different incubation times toward RNA recovery in plasma RNA extraction

RNA in plasma samples are known to exist in marginal amount, contributing to difficulties in obtaining favourable RNA results for downstream applications. Thereby, numerous plasma RNA extraction protocols that utilized different solutions were implemented. These protocols are frequently comparable with slight differences in the parameters especially on the incubation time. Hence, this paper will focus on the implications of different incubation periods in plasma RNA extraction protocol to identify the dependency of plasma RNA towards incubation times and how they are affected. Plasma RNA were extracted using miRNeasy Serum/Plasma (Qiagen, Germany). To test the effects of incubation time, the RNA extractions were repeated with reduced and extended incubation periods for several minutes gaps at certain steps, for instance, the extractions were done distinctly with three- (reduced), five- (default) and seven- minutes (extended) incubation period after TRIzol reagent addition and the results were compared. Our results demonstrated positive correlations when the incubation times were increased during sample homogenization and RNA precipitation, yielding around 14.30 ng/μL to 17.50 ng/μL, and 13.43 ng/μL to 20.05 ng/μL respectively compared to the average of 10.37 ng/μL on default duration. Generally, longer period of incubation might allow sufficient RNA recovery to occur. Steps like sample homogenization and RNA precipitation require longer incubation time for complete RNA recovery since plasma RNA are considerably dignified than DNA or other RNAs due to their low level and encapsulated within exosomes. Thus, the incubation time within extraction protocol play an important role for efficient plasma RNA recovery

Optimization of cell-free plasma RNA extraction for downstream application

The growing interest in biomedical studies has brought RNA from biofluids including plasma, as promising candidates for genetics profiling. The precision and reliability of an analysis in downstream application such as NanoString nCounter® MAX Analysis System (NanoString Technologies, Seattle, WA) ) depend on the RNA quality, purity and level. In this project, NanoString nCounter® miRNA panel was chosen due to rapid identification and ability to profile approximately 800 miRNAs per run which requires total RNAs from plasma with a minimum concentration of 33.3 ng/µL with 260/280 and 260/230 ratios of ≥1.8 for optimal results. Unlike tissues and cells, circulating RNAs in plasma are cell-free and are present in small sizes. However, the abundance of proteins and inhibitors in the plasma as possible contaminants could diminish the effectiveness of molecular isolation techniques and pose challenges in RNA isolation and quantification. This could skew data collection and elucidation. Therefore, the main objective is to determine the optimized plasma RNA isolation protocol to overcome problems in RNA quality and purity with regards NanoString nCounter® MAX Analysis System requirement. Several optimization steps were performed, including the addition of one chloroform extraction step with extra washing steps instead of conducting only once following the actual protocol. After conducting these steps, the average 260/280 ratio falls between 1.7 to 1.8, slightly increased compared to the results before optimization which was around 1.4 to 1.6 since these steps of optimization help to remove excess impurities including phenol and salt. Furthermore, increasing the incubation time in certain steps, for instance, after sample homogenization with Qiazol, during 95% ethanol precipitation and after RNase-free water addition have boosted the RNA recovery allowing RNA concentration of 15 ng/µL and above to be obtained. Hence, the optimized plasma RNA isolation protocol was determined since several issues related to plasma RNA concentration and purity were significantly improved by performing the additional steps in the protocol