Mirna Analysis

The RNAi mechanism (Figure 1) is triggered with the introduction of the small interfering RNA (siRNA) molecules via enzyme (Dicer) breakdown of the double stranded RNA (dsRNA), following an incorporation of these siRNA into an RNA-induced silencing complex (RISC). When the siRNA is

In the brain approximately 70% of the miRNA are expressed and most of it are neuron specific. MiRNA are important element for proper nervous system function. These miRNA plays a crucial role for brain function. Any disruption in the miRNA processing mechanism would cause diseases like ataxia in Purkinje cells, multiple sclerosis in oligodendrocyte cells, Parkinson’s disease in dopaminergic cells, Alzheimer’s disease in α-calcium/calmodulin-dependent protein kinase type II (αCaMKII)-expressing neurons (16).

The development of 19 to 22-nucleotide siRNAs specifically recognizing particular mRNA sequences provides new powerful reagents to selectively down-regulate gene expression and holds great potential not only for the analysis of

RNA isolation for sequencing, qRT –PCR and Ago2-IP for NB was performed, using miRNeasy Mini kit, from Qiagen, according to manufacturer’s instructions, as described in the handbook for

The RNA sequence in the anticodon region, as well as other parts of the transfer RNA molecule, are important

This arrangement provides a convenient mechanism for the coordinated expression of a miRNA and a protein.

The iScript reverse transcription supermix kit for RT-qPCR (Bio-Rad) was used for cDNA synthesis. Master mix was prepared on ice by adding 4 µl 5x RT supermix (iScript RNase H+ MMLV reverse transcriptase, RNase inhibitor, dNTPs, oligo(dT), random primers, buffer, MgCl2, and stabilizers) and 8 µl nuclease-free water for a single reaction. RNA sample was vortexed and 8 µl was added to each PCR tube. Master mix was thoroughly mixed and 16 µl of master mix was added to each PCR tube. PCR tubes containing 20 µl reaction mix were placed in a thermal cycler and reaction protocol was set as follows: Priming for 5 minutes at 25oC, reverse transcription for 30 minutes at 42oC, reverse transcriptase for 5 minutes at 85oC, and hold for a ∞ time at 4oC.

LncRNAs can arise from several different processes; for example, it is possible for local tandem repeat sequences and retro-transposition of non-coding RNA to create lncRNAs (Lee 2012; Clark and Blackshaw 2014). Disruption of coding proteins gene frames can also result in lncRNAs, such as Lnx3 from the Xist gene (Zhu et al. 2013). The formation of new lncRNAs can also arise from the decay of protein coding genes, duplication of other lncRNAs or de novo evolution from previous noncoding sequences, the last of which is the most common (Kapusta and Feschotte 2014). A large proportion of lncRNAs, around 60% in human and murine ES cells, are synthesised from bidirectional transcription at protein coding promoter sites (Kapusta and Feschotte 2014). Other factors that result in the synthesis of lncRNA include transposable elements (TE); in fact, 2 out of 3 lncRNAs transcripts in humans and mice contain at least one TE sequence, which can be found in 20-40% of all lncRNA exonic nucleotides (Kapusta and Feschotte 2014).

We have isolated EVs from the cin1 mutant, CIN1-S isoform mutant, and the WT strains and extracted exRNA. Since the cin1 mutant is drastically reduced in growth, 3x more culture volume than WT was required to obtain an exRNA yield comparable to that of others. exRNA was submitted to the Beijing Genome Institute (BGI, Shenzhen, China) for cDNA synthesis and RNA-Seq. Approximately 100 ng cDNA from each strain was subjected to Illumina deep sequencing using BGISEQ-500 technology. A close to 27 million clean sRNA reads each was obtained from all three strains. The length of sRNA is between 17 nt and 33 nt with most being 21, 22, and 23 nt. The initial sRNA annotation provided by BGI suggested that approximately 96% sRNA sequences were mapped

1998). Interestingly, the phenomenon that the introduction of double-stranded RNAs “quelled” the expression of endogenous genes contain homologous sequences had been observed and even utilized as a tool to knock down gene expression by researchers long before the underlying RNAi mechanism was identified and separated from the antisense silencing by Fire and Mello in 1998 (Fire et al. 1998). They consistently found that the introduction of double-stranded RNAs (dsRNAs) was 100 or more folds effective than the single-stranded “sense” or “antisense” RNAs (ssRNA) to silence the complement gene expression (Fire et al. 1998; Kennerdell and Carthew 1998). Moreover, the dsRNA mediated gene silencing can be transferred between adjacent cells, and even inherited through multiple rounds of cell division (Fire et al. 1998). Therefore, it is proposed that this so called “RNA interference” is an inheritable and transferrable process that involves a catalytic process, which differs from the previously known antisense silencing that ssRNAs directly bind to the complement mRNA targets to repress translation by blocking ribosome access. Subsequently, Tuschl and co-workers demonstrated that RNAi mechanism exists in mammalian cell lines for the introduction of synthetic

In recent years, the next-generation sequencing technology have been used as a powerfull tool by researchers to study cancer data, including cancer diagnosis, therapy, and prognosis. This technology enabled researchers to investigate all human genome transcripts, including non-coding versions. Recent investigations have revealed the association of non-coding RNAs (ncRNAs), including miRNAs and lncRNAs, with cancer pathogenesis, prognosis, survival rate and uses of these ncRNAs as cancer biomarker [21-24].

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals (Brennecke et al, 2005). They consist of approximately 20 to 24 nucleotides of RNA. One process by which miRNAs inhibit gene expression occurs through translational repression (Gu and Kay, 2010). Further to translational repression, endonuclease cleavage can also help repression to occur. According to Cheng et al (2015) ‘dysregulated microRNA expression is correlated with many human diseases’. This suggests that miRNAs can be used as a biomarker for cancer and some diseases such as Cardiovascular Disease (CVD). This is one of the reasons why the scientific community is interested in studying these molecules.

Studies of human and mouse transcriptomes show that protein-coding sequences comprise only a small portion of a genome that is transcribed (Bertone et al.,2004; The ENCODE Project Consortium, 2007; Cheng et al., 2005; Kapranov et al.,2007). RNA has a significant role in gene expression: thousands of noncoding (nc) microRNAs target mRNAs to prohibit expression in about 30 percent of protein coding-genes (Berizilov and Plasterk, 2005). There are many more ncRNAs that are classified as either long or short, some of which have been linked to specific functions. Kim et al. (2010) found that ncRNAs can be directly transcribed from transcriptional enhancer elements. Human cells display significant activity using

On the evolutionary scale biological complexity is heavily increased, without any significant change in the number of protein coding genes whereas non-coding portion of the genome has substantially undergone change. It is now well accepted fact that on increasing evolutionary levels, majority of the genome gives rise to non-coding RNAs (ncRNAs) that led to the increased biological complexity. By halting the central dogma – the flow of information from DNA to RNA to protein, ncRNAs have gained central attention. Recent advancement in the next generation sequencing (NGS) technology allowed discovery of several classes of ncRNAs. lncRNAs are the largest class of ncRNAs, by convention longer than 200 nucleotides in length.

In recent years, the next-generation sequencing technology have been used as an powerfull tool by researchers to study cancer data, including cancer diagnosis, therapy, and prognosis. This technology enabled researchers to investigate all human genome transcripts, including non-coding versions. Recent investigations have revealed the association of non-coding RNAs (ncRNAs), including miRNAs and lncRNAs, with cancer pathogenesis, prognosis, survival rate and uses of these ncRNAs as cancer biomarker[22-25].