Norgen's Total RNA Purification

Recent Work & Current Methods in Immunology microRNA Research

Dr. Bernard Lam Senior Research Scientist Norgen Biotek Corp. [email protected]

Dr. Christoph Eicken Head of Technical Services – Microarrays LC Sciences, LLC [email protected]

Norgen Biotek Corp. www.norgenbiotek.com The Sample Preparation Experts [email protected] microRNAs - What We Know

1. All miRNAs are small non-coding RNAs, usually consisting of ∼20–22 nucleotides for animals and ∼20–24 nt for plants.

2. All miRNA precursors have a well-predicted stem loop hairpin structure, and this fold-back hairpin structure has a low free energy

3. Many miRNAs are evolutionarily conserved, some from worm to human in animals, or from ferns to core eudicots or monocots in plants

4. Bind to complementary mRNA molecules and act as negative regulators of translation

5. High copy number

6. Expression is tissue (and developmental stage) specific

microRNAs - What We Know

1. Currently - 35,828 mature miRNAs across 223 plant, animal, and virus species (miRBase 21, June 2014).

2. Mechanism is far reaching and complex – each miRNA may control many genes and it is estimated that miRNAs regulate expression of up to 50% of all mammalian genes.

3. Operate by one of two hypothesized mechanisms:

– Complete pairing mRNA is degraded - predominant in plants

– Imperfect pairing translation is repressed but mRNA remains intact - predominant in animals Why Study miRNAs in the Immune System?

Ha TY. (2011) Immune Netw 11(1), 11-41. [article] miRNAs in the Immune System: Clinical Potential

Molecular Diagnostics / Biomarkers – Identification of specific miRNAs or miRNA expression based signatures that can act as biomarkers for various diseases/pathologies.

1. Use as biomarkers in body fluids: serum, plasma, exosomes, & HDL particles

2. Make accurate and detailed clinical diagnosis

3. Potential to determine prognosis and predict treatment efficacy

Rosetta Genomics' miRview Lung Assay: Assay Shown to Accurately Differentiate Between the Four Main Types of Lung Cancer miRNAs in the Immune System: Clinical Potential

Drug Discovery / Therapeutics – Identification of miRNAs that play essential roles in disease to act as drugs or possible therapeutic targets.

1. miRNAs as immune system regulating drugs

2. miRNAs as drug targets

3. Study of miRNAs to understand response to infections, stress, other stimuli MicroRNAs in the Circulation (Bodily Fluids)

Circulating Tumor Cells (CTCs)

Schwarzenbach, H. et al. Nat. Rev. Clin. Oncol. 11, 145–156 (2014); [abstract] RNA Purification Technology Consideration for Extracellular Fluids, Vesicles and Single Cell Input

1. Extracellular Fluids (plasma/serum/urine), Extracellular Vesicles (EVs) Low RNA Quantity

2. Specific Cell Types could also be limited in numbers (1 – 10000 Cells)  Low RNA Quantity

3. Small RNAs including microRNAs are commonly found in Extracellular Fluids and Vesicles RNA Purification Technology Must be 1.Sensitive 2.Size Diversity Next Generation Applications has changed the Requirement of Sample Preparation

Phenol:Chloroform

Silica Column Chromatography Most Nucleic Acids (RNA and DNA) Extraction Transcriptome Analysis Products are based on 1. Small Input (CTC, Technologies Developed Exosomes, Liquid Historically (circa 1980’s) Biopsies, Cells) 2. Small RNAs Shortcomings in Diversity and Sensitivity with Traditional RNA Sample Preparation Total Silica

RNA Column

Molecular Cell 46, 893–895, June 29, 2012 Bioanalyzer Loss of GC Diversity: Phenol:Chloroform Extraction Procedures tend RNA on Resolved to have bias in RNA and Loss of Size Diversity and Sensitivity miRNA recovery based on 1. Missing Small RNA including GC contents microRNA and siRNA 2. Require Carrier RNA for Low (Single Cell Input) Norgen Biotek Silicon Carbide as a Matrix for Nucleic Acid Purification for Next Gen Applications

40 Competitor Q No Carrier 35

30 Competitor Q + Carrier 25

20 Ct Value Ct Norgen 15 No Carrier

10 Norgen Competitor A Norgen Competitor Q 1 10 100 1000 10000 100000 1000000 No Phenol Phenol + Silica No Phenol Silica Input Cell Number No Phenol Better Sensitivity: Better Size Diversity: Single Cell RNA Recovery Recover Better Diversity of Small RNA without Carrier Extraction Methods for Total RNA Diversity

Phenol (Trizol) Silica (RNeasy) Silica (miRNeasy) SiC (Norgen) Lysate Lysate Lysate Lysate Preparation Preparation Preparation Preparation 5 minutes 5 minutes 5 minutes 5 minutes Bind RNA Bind RNA 1 minute 1 minute Phase Phase Separation Wash Separation Wash 10 minutes 3 x 1 min. 10 minutes 3 x 1 min. Elute RNA Elute RNA 3 minute Bind RNA 3 minute 1 minute RNA Wash Precipitation ~15 mins ~15 mins 10 minutes RNA without 3 x 1 min. True Total RNA miRNA Elute RNA including RNA Pellet 1 minute miRNA Wash ~25 mins 5 minutes Total RNA 30+ mins Total RNA Single Cell and Sorted Cells RNA Analysis

Norgen Biotek Corp. www.norgenbiotek.com The Sample Preparation Experts [email protected] Sensitivity and Consistency- Reproducibility and Consistency of RNA Isolation is Essential for NGS Applications

40 30 Large RNA S15 miR-21 35

25 R² = 0.992 30

R² = 0.991 Ct Value Ct Ct Value Ct 25 Better 20 20

15 15 Total RNA 1 10 100 1000 10000 100000 1000000 1 10 100 1000 10000 100000 1000000 Input Cell Number Input Cell Number

25 Linear Recovery 5S rRNA Small RNA 20 Isolation R² = 0.998

at Single 15 Ct Value Ct Cell Level 10 of RNA of

5 1 10 100 1000 10000 100000 1000000 All Sizes Input Cell Number

Consistent Recovery of Total RNA from Much higher transcript level Dynmaic Range of Cells (1 – 100000 cells) detected for both large and small Norgen’s Single Cell RNA Purification Kit (#51800) RNA (1 – 100 cells) – 8 uL Elution RNA Isolated from 1 to 100,000 HeLa Cells Norgen’s Single Cell RNA Purification Kit (#51800) Competitor = Arcturus PicoPure RNA Kit Sensitivity and Consistency- Reproducibility and Consistency of RNA Isolation is Essential for NGS Applications

No Carrier RNA Required- NGS Ready

Consistent Recovery of microRNA from Different Cell Lines (10 – 1000 cells) Norgen’s Total RNA Purification Kit (#17200, Identical to miRCURY RNA Kit)

Paggetti et al (CRP-Sante Luxemburg). 2013. Leukemia [abstract] Plasma and Exosomal RNA Analysis

Norgen Biotek Corp. www.norgenbiotek.com The Sample Preparation Experts [email protected]

Small RNA-Seq from as little as 50 μL of Plasma

50 50 4 mL 4

4 mL 4 50 50 ~30nt + Ad

~20nt + Ad NEB Small RNA Library Prep Illumina Small RNA Library Prep Linear RNA Purification Technology Extending Sensitivity of NGS 1. Elution in as little as 10 uL  Bigger fraction of RNA from input plasma used in Library Prep 2. Successful Library Generation using RNA extracted from 50 uL of plasma without any pre-concentration or pre- amplification Plasma/Serum RNA Purification Mini Kit (Cat# 55000)

17 Small RNA-Seq from as little as 50 μL of Plasma

350

50100000 μL vs 4 mL 300 4 mL mL 4 Plasma 200 μL 10000 250 1000 19 200 100

150 from miRNA for reads of # R² = 0.9678 1 1 10 100 1000 10000 100000 100 19 7 # of reads for miRNA from 0.05 mL

100000 #of miRNAs

200mL μL vs 4 mL

10 μL 4 178 10000 50 Elution Plasma

1000 0 9 1 4 100

50 uL 200 uL 4 mL 10

# of reads for miRNA from from miRNA for reads of # R² = 0.9905 50 μL 4 mL 1 1 10 100 1000 10000 100000 # of reads for miRNA from 0.2 mL

NGS on Illumina MiSeq

Linear RNA Purification Technology Extending

Plasma/Serum RNA Purification Mini Kit Sensitivity of NGS (Cat# 55000) Emerging and Challenging Biological Sample Requiring Both Diversity and Sensitivity (Exosomes) Exosomes Isolated by Ultracentrifugation Cellular RNA Norgen Exosome RNA Norgen

Best Yield and Best RIN Value Best Yield and Best Size Diversity of RNA

Eldh et al (Uof Gothenburg). 2012. Molecular Immunology 50:278-286 RNA RNA Isolated from Exosome (Ultracentrifugation) Purification

Norgen’s Total RNA Purification Kit (#17200, Identical to miRCURY RNA Kit) Technology is Competitor = Ambion mirVana, Qiagen miRNeasy, Trizol Critical Simple and Rapid Exosome and Exoxomal RNA Purification Free Circulating RNA

RNA Purification Liquid Biopsies Pure Exosome or (Plasma/Serum/Urine) On-Column/Resin Exosome Lysis Exosome RNA

Norgen

NanoSight Size Distribution

Illumina Small RNA-Seq NanoSight Image

1. Highly Pure Exosome eluted in less than 1 hour 2. Excellent Size Distribution (50 – 100 nm) 3. Complementary RNA Extraction Kit for Next-Generation Expression Studies Better miRNA Diversity Recovered = Better Biomarker Discovery from Clinical Samples

Large Scale NIH Breast Cancer Biomarker Screening involving Serum obtained from 410 individuals

Trizol or Phenol-Silica Kit: An equivalent study will require 410 Phenol:Chloroform Extractions

Godfrey et al (NIH) Breast Cancer Research 2013, 15:R42 Total RNA Purification Kit (#17200) [abstract] microRNA microarrays with 400 uL Serum  Diversity True Compatibility to  Sensitivity and Consistency = Clinical Sample Testing  Simple Workflow  No Phenol Requirement MicroRNA Profiling Review: MicroRNA profiling: approaches and considerations

Colin C. Pritchard, Heather H. Cheng & Muneesh Tewari Nature Reviews Genetics 13, 358-369 (May 2012) .[abstract] Microarray vs RNA Sequencing

Key Advantages of Key Advantages of RNA-Seq Microarray • Provides a comprehensive view of the transcriptome. All transcripts can be • Robust, reliable method, proven over analyzed (mRNA, ncRNA, snoRNA, decades of use lncRNA, miRNA, ...). • High through-put method – 100s of • Not necessarily dependent on any prior samples analyzed per month sequence knowledge. • Streamlined handling – can be easily • Increased dynamic range and tunable automated sensitivity. • Straightforward data analysis • Can detect structural variations such as • Short turn-around time – 5 days gene fusions and alternative splicing events. • Lower cost • A truly digital solution microRNA Microarray Expression Profiling

Differentiated miRNAs of Biological & Statistical Significance - Multiple Chips Biological repeats Control Treated Control Treated

p < 0.05 Array assay

t-Test

p < 0.01

Multi-array normalization and clustering analysis microRNA Microarray Expression Profiling

Sample Data Download & Data Analysis Guide Video: http://www.lcsciences.com/applications/transcriptomics/mirna- profiling/mirna/mirna-example-data/ Microarray vs RNA Sequencing

Comprehensive RNA Sequencing Experiment • Sample QC • Sample preparation • Library preparation • High-throughput sequencing • Advanced bioinformatics analysis • High level customer support Reg. Experimental Design Sample Replicates for Expression Studies Biological Replicates – Still Very Important

• For experiments performed with a small number of biological replicates, significant results may be due to biological diversity between individuals and may not be reproducible - it is impossible to know whether expression patterns are specific to the individuals in the study or are a characteristic of the test condition. • There is no statistical significance for a difference observed between 2 samples. • There is no magic to RNA-Seq. These ideas are widely accepted for DNA microarray experiments, where a large number of biological replicates are now required to justify scientific conclusions.

Hansen KD, Wu Z, Irizarry RA, Leek JT. 2011 Sequencing technology does not eliminate biological variability. Nat Biotechnol 29:572–573. [abstract] Small RNA Sequencing and Data Analysis

Instrument: Illumina HiSeq 2500 Length of Reads: 50 bases Number of Reads: ~100 Million Data Output: ~10-10000 Gb Bar-coding (Indexing) Samples: • We recommend 10-12 samples per lane • The total number of reads does not change with bar-coding • Sacrifice sequencing depth for lower cost Number of Samples Total Reads / Lane Reads/ Sample / Lane 100 M 1 100 M 100 M 2 50 M 100 M 3 33 M 100 M 4 25 M 100 M 5 20 M 100 M 6 16 M Small RNA Sequencing and Data Analysis

Data Flow Raw reads Mappable reads

Reads mapped to Reads un-mapped to mammalian mirs in mammalian mirs in miRBase ACGT101-miR v4.2 miRBase Software

mirs mapped to mirs un-mapped to Reads un-mapped Reads mapped species genome species genome to mRNA, Rfam, to mRNA, Rfam, and repbase and repbase Group 1 others Known species miRNAs

Reads mapped to Reads un-mapped Reads mapped to Reads un-mapped species genome to species genome species genome to species genome

Group Group Group no hit 2 3 4 Known miRNAs Candidate species miRNAs Potentially novel candidate species genome inconsistent with miRNAs miRNAs miRBase Small RNA Sequencing and Data Analysis

Sample Data Download: http://www.lcsciences.com/documents/miRNA-Seq-Sample-Data.zip

Data Analysis Guide Video: http://www.lcsciences.com/news/microrna-sequencing-service-data-analysis-guide/ Breast Milk Exosomes Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes Analyzed miRNA expression profiles in porcine milk exosomes across the entire lactation period by deep sequencing. Found that immune-related miRNAs are present and enriched in breast milk exosomes (p<10−16, χ2 test) and are generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs are present in higher numbers in the colostrums than in mature milk. It was higher in the serum of colostrum-only fed piglets compared with the mature milk- only fed piglets. These immune-related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract.

Gu Y, Li M, Wang T, Liang Y, Zhong Z, Wang X, Zhou Q, Thirteen well-characterized immune-related miRNAs. The data Chen L, Lang Q, He Z, Chen X, Gong J, Gao X, Li X, Lv are normally distributed (Kolmogorov-Smirnov test, p>0.05). X. (2012) Lactation-Related MicroRNA Expression The statistical significance was calculated by Student’s t-test (n Profiles of Porcine Breast Milk Exosomes. PLoS = 6 per group). Values are means±SD. CF: colostrum-only ONE 7(8): e43691. [article] feeding; MF: mature milk-only feeding. Innate Immune Response Regulation microRNA 181 inhibits porcine reproductive and respiratory syndrome virus replication and has implications for controlling virus infection Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in swine industry.

Report that miR-181 can directly impair PRRSV infection. Results showed that delivered miR-181 mimics can strongly inhibit PRRSV replication in vitro through specifically binding to a highly conserved region (over 96%) in the downstream of miR-181c exhibits antiviral activity in vivo and relieves pigs from PRRSV- open reading frame 4 (ORF4) of the induced fever. (A) Rectal temperature curve of pigs from two groups after viral genomic RNA. The inhibition of PRRSV JXwn06 infection. (B) Distribution of average rectal temperatures. PRRSV replication was specific and (C) Viral growth curves in pigs of two groups. (D) Analysis of viral load in serum from 3 individuals with control (E) Survival curves of pigs treated dose-dependent. with miR-181c or NC mimics after PRRSV JXwn06 infection. Guo XK et al. (2013) Increasing expression of microRNA 181 inhibits porcine reproductive and respiratory syndrome virus replication and has implications for controlling virus infection. J Virol 87(2), 1159-71. [abstract] Innate Immune Response Regulation microRNA 181 inhibits porcine reproductive and respiratory syndrome virus replication and has implications for controlling virus infection Highly pathogenic PRRSV (HP- PRRSV) strain-infected pigs treated with miR-181 mimics showed substantially decreased viral load in blood and relief from PRRSV- induced fever compared to negative control (NC)-treated controls.

These results implicate the important role of host miRNAs in modulating PRRSV infection and viral pathogenesis, and also support miR-181c exhibits antiviral activity in vivo and relieves pigs from PRRSV- the idea that host miRNAs could be induced fever. (A) Rectal temperature curve of pigs from two groups after useful for RNAi-mediated antiviral PRRSV JXwn06 infection. (B) Distribution of average rectal temperatures. therapeutic strategies. (C) Viral growth curves in pigs of two groups. (D) Analysis of viral load in serum from 3 individuals with control (E) Survival curves of pigs treated with miR-181c or NC mimics after PRRSV JXwn06 infection. Guo XK et al. (2013) Increasing expression of microRNA 181 inhibits porcine reproductive and respiratory syndrome virus replication and has implications for controlling virus infection. J Virol 87(2), 1159-71. [abstract] Virus - host interaction

In-depth profiling and analysis of host and viral reveal involvement of microRNAs in host-virus interaction in teleost fish MirNA-Seq used to identify and analyze both host and viral miRNAs in Japanese flounder (Paralichthys olivaceus), an economically important teleost fish, infected with megalocytivirus at a timescale of 14 days divided into five different time points. Results showed that a total of 381 host miRNAs and 9 viral miRNAs were identified, the latter being all novel miRNAs that have no homologues in the currently available databases. Of the host miRNAs, 251 have been reported previously in flounder and other species, and 130 were discovered for the first time.

Zhang et al. (2014) In-depth profiling and analysis of host and viral microRNAs in Japanaese flounder infected with megalocytivirus reveal involvement of microRNAs in host-virus interaction in teleost fish. BMC Genomics 15:878 [article] Virus - host interaction

In-depth profiling and analysis of host and viral reveal involvement of microRNAs in host-virus interaction in teleost fish

The expression levels of 121 host miRNAs were significantly altered post-viral infection (pi), and these miRNAs were therefore classified as differentially expressed host miRNAs.

The expression levels of all 9 viral miRNAs increased from 0 d pi to 10 d pi and then dropped from 10 d pi to 14 d pi.

In-depth profiling and analysis of host and viral reveal involvement of microRNAs in host-virus interaction in teleost fish For the 121 differentially expressed host miRNAs and the 9 viral miRNAs, 243 and 48 putative target genes, respectively, were predicted in flounder. GO and KEGG enrichment analysis revealed that the putative target genes of both host and viral miRNAs were grouped mainly into the categories of immune response, signal transduction, and apoptotic process.

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Norgen Biotek Corp. www.norgenbiotek.com The Sample Preparation Experts [email protected]