Recommended
Recommended
More Related Content
What's hot
What's hot (20)
Similar to miRNA Biogenesis, Isolation, Quantification Methods
Similar to miRNA Biogenesis, Isolation, Quantification Methods (20)
More from SAIMA BARKI
More from SAIMA BARKI (7)
Recently uploaded
Recently uploaded (20)
miRNA Biogenesis, Isolation, Quantification Methods
- 1. miRNA: Biogenesis, Mechanism of Action, Isolation Protocol, and Quantification Methods SAIMA BARKI CELL BIOLOGY LAB QUAID-e-AZMA UNIVERSITY ISLAMABAD, PAKISTAN 19-03-2019
- 2. INTRODUCTION SMALL Functional, posttranscription and translational regulation Noncoding Pre-mirna=75 nts,Mature 18-25 nts, Highly conserved Founding gene-lin-4 and let-7-3’ UTR complementary Imperfect Watson- crick base pairing Relativley long life, High level of RNAses in biofluids Complexed with biopolymers
- 3. NOMENCLATURE
- 11. Spectrophotometric analysis, Quantification cycle (Ct) mean, PCR efficiency and correlation-coefficient (R2) values of miR-21
- 13. q-PCR amplification profiles of miR-21 using KCH3COOH method.
- 14. Efficiency of methods to precipitate large RNAs and final RNA isolation protocol
- 16. miRNA Target identification Bioinformatic tools Immunoprecipitation study Omic based study
- 17. Stem-loop Primer/Polyadenylation/s- poly(oilgodT) based Reverse transcription
- 19. Stem-loop RT method vs. Poly(A) method
- 20. stem–loop primers might provide better RT efficiency and specificity than linear.
- 21. S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer
- 22. Dynamic range and sensitivity of the S-Poly(T) method used in miRNA quantification
- 23. Comparison of the sensitivity of three different miRNA qPCR assays
- 25. The Ct values of miRNA assays using three different methods
- 26. Specificity of the S-Poly(T) method in human let-7 family miRNAs assay.
- 28. miRNAs profiling between hypoxic and normoxic HPASMC
- 30. Expression profiling of human serum miRNAs
- 31. Conclusion: Specific miRNA isolation kit from different clinical samples or opting single optimized protocol for different clinical samples. The choice of sample for miRNA should be done in accordance to the available kits and protocols and the requirement of the research. S-poly(oligodT) primer based RT reaction give the accurate, specific, sensitive results. miRNA play an important role in different pathological conditions and so it is a novel molecular marker for diagnosis and also novel therapeutic strategy.
Editor's Notes
- Possible mechanisms for miRNA gene regulation. Unregulated mRNAs engage with the initiation factor eIF4F complex, which is composed of eIF4A, eIF4E and eIF4G subunits and recruits ribosomal subunits, which form circularized structures that enhance translation (upper left). When miRISC binds to target mRNAs, a high degree of miRNA–mRNA complementarity facilitates Ago-catalyzed degradation of target mRNA sequences through mRNA cleavage mechanisms (lower left). Alternatively, central mismatches prevent degradation and facilitate translational repression by any of four (a–d) possible mechanisms (right): (a) miRISCs bind to target mRNAs and represses initiation at the cap recognition stage, or at (b) the 60S ribosomal recruitment stage, (c) miRISC can prevent mRNA to circularize (d) miRISC attachment to target mRNAs also facilitates premature separation from ribosomes, which represses translation at the postinitiation stag
- Choice of samples. A, Workflow for the preparation of platelet-poor plasma (PPP) from platelet-rich plasma (PRP). Addition of prostacyclin inhibits platelet activation during centrifugation. B, Concerns with regards to miRNA measurements in the different samples range from a contamination with leukocytes in PRP, proteolytic activity in serum to residual platelets in plasma. PRP and serum should reflect platelet miRNA content; PPP platelet miRNA release; plasma samples will reflect extracellular miRNA content but may additionally reflect either, platelet miRNA content or release. This depends on the amount of residual platelets in the plasma samples or platelet activation during plasma preparation, in particular during centrifugation.
- Samples were precipitated (200 μL) with both method 1 (KCH3COOH and final precipitation with 2.5 M LiCl plus ethanol) and method 2 (2.5 M LiCl and ethanol directly after chloroform treatmen
- Q-PCR amplifcation plot of amplicon generated using mature miR-21 isolated from (A) TH-29 cell line, (B) plasma, and (C) urine cells with different methods.
- Q-PCR amplifcation profles of miR-21 using KCH3COOH method. (A) The amounts of cDNA used for q-PCR with different dilution factors as follows: 1 (1 μg); 2 (10-1 μg); 3 (10-2 μg); 4 (10-3 μg); 5 (10-4 μg); 6 (negative control); 7 (water control). (B) Melting curve. (C) Standard plot.
- Nucleic acid reagents used for and intermediate products generated in this method. A, Mature miRNA (blue). A-H, Light blue lines show the boundary of the mature miRNA sequence within the RT and qPCR reagent sequences; open arrowheads indicate directions of polymerization. B, The stem-loop primer 5′ 6 nt annealed with mature miRNA 3′ 6 nt; RT, reverse transcriptase. C and D, First strand cDNA, after polymerization, C, and heat denaturation, D. E, Forward primer with added 5′ nts. F, Second strand cDNA. G, Hydrolysis probe and reverse primer. H, PCR product defined by the 5′ termini of the forward and reverse primers.
- Specificity of TaqMan miRNA assays between stem–loop and linear RT primers. Mature let-7a-specific assay was tested against let-7a, let-7e and pri-miR precursor let-7a-3. DCT represents the CT difference between two targets or methods. A total of 1.5 · 108 copies of synthetic targets were added to each RT reaction.
- Dynamic range and sensitivity of the S-Poly(T) method used in miRNA quantification. A: Correlation of total RNA input to the threshold cycle (Ct) value. Five miRNAs including hsa-miR-92a, hsa-miR-210, hsa-miR-16, hsa-miR-21 and hsa-miR-223 were assayed by the S-Poly(T) method with a series of 10-fold diluted total RNAs prepared from HEK293 cells. The amount of total RNA in each qPCR reaction was ranged from 10 ng to 0.01 pg. The amplification efficiency was 96% (miR-92a), 95% (miR-210), 91% (miR-16), 90% (miR-90), and 101% (miR-223), respectively, and the correlation coefficients (R2) for each miRNAs was .0.99. B: Amplification plot of hsa-miR-92a over seven orders of magnitude. NTC: no-template control; -RT: no-reverse transcriptase control. All PCR reactions were performed in triplicate.
- *: miR-21, miR-16 and miR-210 were assayed with SYBR Green I, while miR-140-5p was assayed with universal (S-Poly(T)) or specific (stem-loop) Taqman probe. doi:10.1371/journal.pone.0048536.t001
- A: Eight hsa-let-7 miRNAs (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i) were individually over-expressed in HEK293 cells by a lentiviral vector. The expression levels of the eight hsa-let-7 miRNAs were compared between the control (transduced with blank lentiviral vector) and the hsa-let-7 miRNAs-overexpressing HEK293 cells. The expression level of each hsa-let-7 miRNA in the hsa-let-7 miRNAs-overexpressing HEK293 cells (OE-7a,OE-7i) was shown as the fold change compared with that in the control (Con). B: sequence alignment of eight hsa-let-7 miRNAs. Non-conserved nucleotides are shaded. C: discrimination of mature miRNA from pri- and/or pre- miRNA by the S-Poly(T) and Poly(A) methods. The total RNA overexpressing hsa-let-7b and hsa-let-7c were reverse transcribed to cDNA by the S-Poly(T) and Poly(A) methods, respectively. The PCR products were run on 4% agarose gel in sodium borate (SB) buffer. The arrows indicate the amplification products of pri- and mature miRNAs. doi:10.1371/journal.pone.0048536.g004
- MiRNAs profiling between hypoxic and normoxic HPASMC. A, B: The accuracy for quantification of miR-199a-3p (A) and miR-210 (B) was evaluated in a high-throughput RT reaction. The number of pooled RT primers in a single RT reaction ranged from 1,360. C: Scatter plot of relative expression level of 721 miRNAs determined by S-Poly(T) qPCR. The relative expression of miRNA was calculated as 2‘-DCt and presented in the logarithmic scale. The data on the line across both X and Y axes represented the constitutively expressed miRNAs. D, E: Significantly up-regulated and down-regulated miRNAs. The expression of miRNAs was shown as fold change between hypoxia and normoxia. F: Scatter plot of the relative expression level of 246 miRNAs detected by LNA-based miRNA microarray. The relative expression of miRNA was calculated as Hy3/Hy5 ratio and the data on the line across both X and Y axes represented the constitutively expressed miRNAs. G: Heat map showing the expression of miRNAs that are differentially expressed in hypoxic HPASMC compared with normoxic HPASMC. Red color denotes induction and blue color denotes suppression. H: Significantly up-regulated miRNAs verified by real-time PCR. The expression of miRNAs was shown as fold change between hypoxia and normoxia. Each bar represents mean 6 SD. Statistical s
- A: Pooled total RNAs from 40 healthy human serum samples were used to assay human 1,080 miRNAs in the miRBase 16.0. A scatter plot of 518 human serum miRNAs with Ct ,35 assayed by S-Poly(T) method was shown. Arrows indicate the most abundant miRNAs. B: Box-whisker plots of the three most abundant miRNAs, namely miR-648, miR-4298 and miR-16 in each healthy serum sample (n = 40). The boxes indicate the 75th and 25th percentiles. The whiskers denote the lowest and highest values. The lines inside the boxes indicate the median values. doi:10.1371/journal.pone.0048536.g006