It s All in the Details (or Small RNA): Simplified and Improved mirna Purification from Tissue

Transcription

1 It s All in the Details (or Small RNA): Simplified and Improved mirna Purification from Tissue Douglas Horejsh January 2015

2 Discussion Outline Non-coding RNA General Overview mirna What is it? The Future of mirna Downstream Assays Purification of mirna on the Maxwell RSC Instrument 2

3 Humans Have an Abundance of Non-coding RNA Compared with Other Species Organism Percent of Transcriptional Output Protein coding RNA Non-coding RNA E. coli S. cerevisiae C. elegans D. melanogaster H. sapiens 2 98 Higher organisms with more complex regulation mechanisms have an abundance of non-coding RNA 3

4 Non-coding RNA Seven Major Non-coding RNA Families Ribosomal RNA (rrna) Transfer RNA (trna) Small nuclear RNA (snrna) Small nucleolar RNA (snorna) Long non-coding RNA (lncrna) Short interfering RNA (sirna) MicroRNA (mirna) 4

5 Non-coding RNA Each Type of ncrna Functions Differently 5

6 mirna Overview Post-transcriptional Regulation of Gene Expression mirnas nucleotides in length Found in plants, animals and viruses One mirna can target and regulate multiple mrnas Many mirnas can bind to the same mrna target Up or down regulate gene expression Involved in biological processes which impact human health and disease Positively or negatively impact human health and disease 6

7 MicroRNA Biogenesis It s a Complicated Cellular Process Nucleus mirna gene is transcribed by RNA Pol II Drosha/DGCR8 proteins crop the pri-mirna to pre-mirna EXP5/RAN*GTP involved in export into the cytoplasm Cytoplasm Dicer/TRBP cleaves pre-mirna to release mature mirna Pre-RISC formed by loading of AGO protein Mature RISC formed with unwinding of mature mirna The mirna is now available for binding to mrna targets 7

8 What Happens After Biogenesis? mirna Regulate Target mrna Expression mirna-mediated repression of target mrna RBP modulation of mirna binding results in loss of mirna-mediated repression Molecular Oncology 6 (2012), p

9 mirna in Cancer Multiple mirna Species Have Been Linked to Cancer Normal tissues Mature mirna binds to mrna to block translation resulting in normal rate of growth, proliferation and cell death mirna acting as tumor suppressor Loss of mirna binding to oncogene target leads to more translation of the oncogene, resulting in tumor formation mirna functioning as an oncogene High level of mirna binding to tumor suppressor target leads to inhibition of tumor suppressor transcript translation, resulting in tumor formation Nature Reviews Cancer 6 (2006), p.263 9

10 Direct Interaction with Tumor Supressor p53 mrna Very complicated process Interest in biomarker discovery Molecular Oncology 6 (2012), p

11 The Future of mirna Move from research to diagnostic More complexity must be unraveled More biomarkers must be identified Change from invasive testing to liquid biopsy 11

12 Multiple Downstream Assays Are Used to Assess mirna Levels in Cells RT-qPCR Target specific priming Universal priming Microarrays Next-gen sequencing 12

13 Two Reasons Not to Use a Gel or Bioanalyzer to Assess mirna Levels and Quality RNA Quality Assessment The mirna fraction is such a small component of total RNA that it is not very prominent The presence of mirna and other small RNA species alter the RIN 13

14 Target Specific Priming of mirna Stem-loop primer is used for the RT step Laborious workflow as individual RT reactions needed for individual qpcr 10ng of total RNA input into RT Small dilution of cdna used for qpcr Typical qpcr with forward primer against mirna and reverse primer against stem-loop sequence Probe sits on remainder of mirna seq and into the stem sequence 14

15 Universal Priming: Assess mirna Levels Without Setting Up Individual RT Reactions As the (1) RT/ (1) PCR specific approach is tedious, other groups have developed a universal RT approach The universal primer enables detection of any mirna in the cdna sample Typically only works with dye intercalators 15

16 Next-Gen Sequencing The Newer Kid on the Block Unbiased quantitation that does not depend on the previous identification of each mirna species; enables discovery of new mirnas Purple.emz 16

17 Next-Gen Sequencing of mirna Process (con t.) Purple.emz 17

18 Maxwell RSC Instrument Flexible, Personal Automated Nucleic Acid Purification Reliable Prefilled cartridges and preprogrammed methods means consistent purification. Flexible Optimized kits available for DNA or RNA purification from a variety of sample types including blood, cells and tissue (fresh and frozen). Intuitive Minimal training required. The instrument software guides users through a purification run. Efficient Automated nucleic acid purification frees up your time, and the integrated Quantus Fluorometer streamlines the next quantitation step. 18

19 Plastic Plungers Protect Against Cross-contamination A magnetic bar assembly covered by a plastic sheath (plunger), moves the paramagnetic particles from well to well. Magnets are positioned at the tips of the 16 vertical rods Magnetic Bar Plunger Elution Tube (30-100µl) 19

20 It s a Magnetic Particle Mover Maxwell RSC Moves the Particles Not the Liquid Samples are processed using prefilled reagent cartridges, simplifying instrument setup and providing consistently high yields and purity. Because there is no liquid handling, there are: No drips No clogs Fewer breakdowns No detectable cross contamination Elution Tube (30-100µl) 20

21 Maxwell RSC Approach for mirna Purification Total RNA Including mirna Extraction without Organics Initial offering for mirna purification from tissue This challenging sample type as the initial kit will allow for processing from other samples with little adaptation Extracts Total RNA including mirna Total RNA allows researchers to choose their best controls and normalize to mrna if desired Additional RNA purification no longer necessary from precious samples No organics used Safer and just as effective 21

22 Maxwell RSC Approach for mirna Purification A Simple Protocol with Minimal Pre-processing 20mg tissue Homogenize in 200µl Homogenization Buffer + thioglycerol Add 200µl Lysis Buffer + proteinase K & incubate 10 minutes Preprocessing Automated Purification Analysis 20 minutes 80 minutes Sequencing, arrays, RT-PCR, RT-qPCR 22

23 Maxwell mrna Purification Kit High Quality mirna from Tissue Very simple pre-processing before automated extraction Adaptation of simplyrna tissue chemistry Optimized instrument method Works well in next-gen sequencing Early access researchers have worked with the chemistry yielding great results 23

24 Seven Major Performance Criteria Examined During Kit Development 1. RNA Purity 2. RNA Quantity 3. Good mirna yield from: 1mg Input 10mg Input 4. No Cross-contamination between samples 5. Good message (mrna) yield from: 1mg Input 10mg Input 6. Minimal DNA contamination in purified mirna 7. Minimal RT-qPCR inhibitors in purified mirna 24

25 Absorbance Ratio High Purity Ratios of Purified RNA Both A 260 /A 280 & A 260 /A 230 Greater Than / / Liver Kidney Pancreas 25

26 Concentration (ng/µl) High Yields of Concentrated Total RNA Total RNA Concentration and Yield by Absorbance µg µg µg Liver Kidney Pancreas 26

27 Yield in copies Yield in copies Testing Levels of Different mirnas by RT-qPCR 1mg Tissue Input Per Purification 6.E+08 1mg Input for Let-7a RT-qPCR Amplification 6.E+08 1mg Input for mir-21 RTqPCR Amplification 5.E+08 5.E+08 4.E+08 4.E+08 3.E+08 3.E+08 2.E+08 2.E+08 1.E+08 1.E+08 0.E+00 Pancreas Kidney Liver 0.E+00 Kidney Liver Pancreas 27

28 Yield in copies Yield in copies Testing Levels of Different mirnas by RT-qPCR 10mg Tissue Input Per Purification 10mg Input for Let-7a RTqPCR Amplification 10mg Input for mir-21 RTqPCR Amplification 1.E+10 1.E+10 8.E+09 8.E+09 6.E+09 6.E+09 4.E+09 4.E+09 2.E+09 2.E+09 0.E+00 Pancreas Kidney Liver 0.E+00 Kidney Liver Pancreas 28

29 No Cross-contamination Detected Across 16 Samples Cartridge layout on the deck of the Maxwell RSC instrument water blank mirna tissue sample water blank mirna tissue sample water blank mirna tissue sample water blank mirna tissue sample water blank mirna tissue sample water blank mirna tissue sample water blank mirna tissue sample water blank mirna tissue sample Alternating wells of samples (Negative Control-Water followed by Positive Control mirna tissue sample) Tested all purified samples for the presence of mirna by RT-qPCR Results: All water wells showed signal below lowest standard, if any at all All sample wells showed high levels (about 2x10 9 copies) of mirna 29

30 mrna Amplification by RT-qPCR 1mg Tissue Input Tissue Input (1mg) Ct Value Conc. (ng/µl) Mouse Kidney microglobulin mrna target Mouse Liver Mouse Pancreas Good, consistent amplification across experimental replicates 30

31 mrna Amplification by RT-qPCR 10mg Tissue Input Tissue Input (10mg) Ct Value Conc. (ng/µl) Mouse Kidney microglobulin mrna target Mouse Liver Mouse Pancreas Good, consistent amplification across experimental replicates 31

32 Minimal DNA Contamination Detected Tissue Input (10mg) DNA Conc. (ng/µl) RNA Conc. (ng/µl) % DNA Mouse Liver Mouse Liver Mouse Liver Mouse Liver

33 Minimal RT-qPcR Inhibition Observed in Eluates The Solaris RT-qPCR inhibition assay was used to test inhibition An increase in Ct of <2 Ct is not inhibited, 2-3 Ct is somewhat inhibited, and >3 is inhibited Input Type Ave. Ct Value Mean Delta Ct Kidney Liver Pancreas No Inhibition Control N/A IPC Block (10% SDS) No Ct N/A Samples all had less than 2 Ct shift, indicating no inhibition 33

34 Kit Development Summary Matched All Required Performance Criteria Criteria Criteria Met 1. RNA Purity Yes 2. RNA Quantity Yes 3. Sufficient mirna from 1-10mg tissue Yes 4. No Cross-contamination between samples Yes 5. Sufficient message (mrna) from 1-10mg tissue Yes 6. Minimal DNA contamination in purified mirna Yes 7. Minimal RT-qPCR inhibitors in purified mirna Yes 34

35 Additional Performance Testing Extracted mirna Works in NGS 35

36 Log2 Maxwell mirna Purification Performs Favorably vs. Competitor with More Complete Target Diversity 2.00 Liver, low expressing targets (<7330 copies, >10 copies), n=521 Liver, fewer than 10 copies Detected only with Maxwell 980 different mirna Detected only with Competitor 339 different mirna Maxwell Competitor # raw reads 10,449,341 8,908,461 # of reads containing 3ADT 10,373,707 8,786,473 # reads <15 nt after removing 3ADT 755, ,052 # reads after removing 3ADT 9,618,102 8,428,421 # mappable reads 9,594,870 8,409,869 The Maxwell mirna purification yielded more sequence diversity in low expressing mirna 36

37 Quotes from Customers that Tested the Maxwell mirna Kit The yield received from the kit was stunning. We were really thrilled with the amount and purity. Acceptable yield. Purity (260/230 is 2.0). Quick. mir RT-PCR worked. Automated, still as simple as simplyrna, can afford kits same price as competitor. Cleaner, more consistent samples with higher yield over manual. 37

38 Conclusions mirna are short RNA of about nts Very important in gene regulation and cancer Most mirna studies are focused on basic research and discovery, but diagnostic use may explode over the next 3 years Promega has developed a mirna purification kit that extracts total RNA including mirna on the Maxwell RSC Instrument High yields and purity Minimal DNA contamination or RT-qPCR inhibitors in the purified RNA The kit will be available in Q1 of

39 Questions? 39