The Hows and Whys of Early Steps in RNA Analysis

Transcription

1 The Hows and Whys of Early Steps in RNA Analysis Leta Steffen, PhD Sr Research Scientist, Promega Promega Corporation

2 Presentation Outline Optimizing the Early Steps Improves Analysis RNA Analysis Workflow Cells and FFPE samples End - point PCR Extraction/ Purification Protecting RNA from degradation Quantitation Reverse Transcription Analysis Purify Purify Quantify Protect Reverse Transcribe Northern Blot qpcr Microarray NGS Options at each step Quality control The MIQE Guidelines Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Bustin et al. (2009). Promega Corporation 2

3 RNA Analysis Workflow Northern Blot End - point PCR Purify Quantify qpcr Protect Microarray Reverse Transcribe Next - Gen Sequencing Promega Corporation 3

4 Purification Challenges Extracting Intact RNA with High Yield and Purity Purification Challenges Purifying sufficient RNA from: Small, precious samples Degraded (FFPE) Purify Maintaining RNA integrity Degraded (FFPE) Isolating pure RNA No gdna contamination No inhibitors Promega Corporation 4

5 RNA Purification Chemistries Organic Extraction Phenol, Trizol Phenol inactivates RNases Inexpensive Manual method, variable between scientists Hazardous chemicals Spin column RNA capture on membranes RNase inactivated with guanidinium thiocyanate buffer Variety of membrane materials Less variable outcomes Manual method with higher throughput, can be automated Bead-based RNA capture on magnetic beads RNase inactivated with guanidinium thiocyanate buffer Variety of membrane materials Automation friendly but can be setup for manual Promega Corporation 5

6 RNA Purification Chemistries Organic Extraction Phenol, Trizol Phenol inactivates RNases Inexpensive Manual method, variable between scientists Hazardous chemicals Spin column RNA capture on membranes ReliaPrep Kits RNase inactivated with guanidinium thiocyanate buffer Variety of membrane materials Less variable outcomes Manual method with higher throughput, can be automated Bead-based RNA capture on magnetic beads Maxwell System RNase inactivated with guanidinium thiocyanate buffer Variety of membrane materials Automation friendly but can be setup for manual Promega Corporation 6

7 RNA Purification Chemistries Contaminants in Each Method Contaminants Organic Extraction Spin column Bead-based Genomic DNA X X X Phenol X Alcohol X X X Guanidinium X X Promega Corporation 7

8 RNA Purification Protocols Purification from Cells Purification from FFPE tissues 1. Sample Prep: i. Lyse cell pellet (100-5x10 6 ) in Lysis Buffer 2. Binding: Bind RNA to binding column or beads; DNase I 3. Washing: To remove impurities 4. Elution: Elute purified RNA 1. Sample Prep: i. Deparaffinize ii. ProK digest iii. De-crosslink iv. DNase I 2. Binding: Bind RNA to the binding Column or beads 3. Washing: To remove impurities 4. Elution: Elute purified RNA in low volumes Promega Corporation 8

9 Purification from FFPE A fast, simplified workflow with Promega FFPE RNA kits Step Traditional Protocols Promega Protocols De-paraffinize Xylenes or other organics Mineral oil + heat Lyse/De-crosslink Proteinase K + heat Proteinase K + heat Purify nucleic acid Phase extraction (phenol chloroform) DNase treatment Capture on resin or membrane Remove salts etc. Precipitation & alcohol wash Alcohol wash Recover nucleic acid Remove contaminating nucleic acids Precipitation/Rehydration DNase treatment Time 2 days 2-4 hours Simple particle or column elution Promega Corporation 9

10 Purification Challenges Extracting Intact RNA with High Yield and Purity Purification Challenges Purifying sufficient RNA from: Small, precious samples Degraded (FFPE) Purify Maintaining RNA integrity Degraded (FFPE) Isolating pure RNA No gdna contamination No inhibitors Promega Corporation 10

11 RNA Purification Chemistries High yield and high purity Isolation from 100 Cells Purification of RNA with No RT-PCR Inhibitors qpcr Input Volume 1µl 5µl 9.5µl RNeasy Mini (30µl Elution) 100% 94% 85% ReliaPrep Cell (30µl Elution) 100% 105% 109% ReliaPrep Cell (15µl Elution) 100% 115% 117% Promega Corporation 11

12 Average Cq (n = 3) RNA Purification Chemistries gdna contamination in RNA Amplification of Human 2-microglobulin from Equal Volumes of Eluate Maxwell 16/simplyRNA Cell Kit QiaCube/RNeasy Cell Kit TRIzol/Manual Low gdna content (High Cq) gdna contamination Cq difference means fold more gdna in these preps ,000K 100K 10K 1K No RT (1,000K) HEK 293 Cells/Isolation No RT Promega Corporation 12

13 RNA Analysis Workflow Northern Blot End - point PCR Purify Quantify qpcr Protect Microarray Reverse Transcribe Next - Gen Sequencing Promega Corporation 13

14 RNA Protection RNA s Worst Enemies RNA self-hydrolysis Due to additional free OH group Increased with Higher ph Cations, metals Temperature Freeze-thaw Ribonuclease (RNase) Degrade precious RNA samples Ubiquitous Difficult to permanently inactivate Do not require cation cofactors Surprisingly common in lab chemicals Bovine Ribonuclease A Conserved sequence in gray (Chelcie H. Eller et al. J. Biol. Chem. 2014;289: ) Promega Corporation 14

15 RNA Protection Ribonuclease in common lab chemicals RNA was incubated with common laboratory reagents overnight with and without RNasin RNase contamination degraded samples in this RNA laboratory! Despite RNase-free label! RNasin Ribonuclease protected Promega Corporation 15

16 Protecting RNA from Degradation Handle samples carefully Wear gloves, use disposable nuclease-free plastics Clean surfaces (RNaseZAP, ELIMINASE ) Use dedicated spaces & pipets Store samples in appropriate buffers Buy well characterized reagents! Nuclease-free water, DEPC-treated water Acidic conditions, no cations/metals Store purified RNA at 4 C or freeze in single-use aliquots Protect your RNA Use RNasin Ribonuclease inhibitors downstream applications RNase Contamination Happens; Recombinant RNasin Inhibitor Can Safeguard Your Samples. Hendricksen A, Hook B, and Schagat T. Promega Corporation 16

17 RNA Analysis Workflow Northern Blot End - point PCR Purify Quantify qpcr Protect Microarray Reverse Transcribe Next - Gen Sequencing Promega Corporation 17

18 Total RNA Quantification and Quality Assessment Key Challenges Accurately measuring small RNA amounts Assessing RNA integrity Determining RNA Purity - Chemical carryover - DNA Contamination Promega Corporation 18

19 Total RNA Quantification and Quality Assessment Gel Electrophoresis Agilent 2100 Bioanalyzer Accurately measuring small RNA amounts UV Absorbance Spectrophotometer NanoDrop /NanoVue Assessing RNA integrity Fluorescent Dye-based RT-qPCR Determining RNA Purity - Chemical carryover - DNA Contamination Promega Corporation 19

20 Gel Electrophoresis Agarose and acrylamide Denaturation required (formamide, glyoxal) RNA molecules separated on the basis of size RNA stained with a fluorescent RNA binding dye Ethidium bromide Diamond Dye, SYBR Green II, and SYBR Gold RNA quantification Estimate the relative intensity of fluorescence Gel densitometry Promega Corporation 20

21 Total RNA Quantification and Quality Assessment Gel Electrophoresis Accurately measuring small RNA amounts Measurement is qualitative Gel densitometry based quantitation Type of gel & dye affects sensitivity Large sample required gdna contamination 28s rrna 18s rrna degraded RNA Promega Corporation 21

22 Total RNA Quantification and Quality Assessment Gel Electrophoresis Assessing RNA integrity Expect 2:1 ratio of 28S:18S rrna Qualitative assessment RNA from cell culture should look ideal RNA from FFPE looks degraded gdna contamination 28s rrna 18s rrna degraded RNA Promega Corporation 22

23 Total RNA Quantification and Quality Assessment Gel Electrophoresis Determining RNA Purity - Chemical carryover - DNA Contamination Genomic DNA contamination visible Does not give information on other contaminants gdna contamination 28s rrna 18s rrna degraded RNA Promega Corporation 23

24 Agilent 2100 Bioanalyzer Microfluidics chip 1 μl input Low hands-on time, 12 samples per chip, ~40 minutes per run Automated & quantitative analysis Electropherograms & gel-like images RNA concentration RNA Integrity Number (RIN) 28S : 16S rrna ratio Promega Corporation 24

25 Total RNA Quantification and Quality Assessment Agilent 2100 Bioanalyzer Accurately measuring small RNA amounts HEK293 Cells RIN = S rrna 18S rrna Marker Quantitative measurement relative to a standard 1μL sample required High sensitivity Nano LabChip ng/μl Pico LabChip pg/μl FFPE Sample RIN = NA Promega Corporation 25

26 Total RNA Quantification and Quality Assessment Agilent 2100 Bioanalyzer Assessing RNA integrity HEK293 Cells RIN = S rrna 18S rrna Marker Analysis is automated Analysis is quantitative RIN value represents RNA integrity Useful for cell culture Rarely useful for FFPE FFPE Sample RIN = NA Promega Corporation 26

27 Total RNA Quantification and Quality Assessment Agilent 2100 Bioanalyzer Determining RNA Purity - Organics carryover - DNA Contamination HEK293 Cells RIN = S rrna 18S rrna Does not give information on RNA purity Genomic DNA is too large to be assessed Not predictive of downstream success Marker FFPE Sample RIN = NA Promega Corporation 27

28 Total RNA Quantification and Quality Assessment Gel Electrophoresis Agilent 2100 Bioanalyzer Accurately measuring small RNA amounts UV Absorbance Spectrophotometer NanoDrop /NanoVue Assessing RNA integrity Fluorescent Dye-based RT-qPCR Determining RNA Purity - Chemical carryover - DNA Contamination Promega Corporation 28

29 UV Absorbance Each Wavelength Measures Different Components Output Wavelength 260nm 280nm Measurement Nucleic acids A 260 of 1.0 = 50µg/mL for dsdna 40µg/mL for RNA 33µg/mL for ssdna Protein 230nm Guanidinium, phenol, EDTA, protein 320nm Background scattering Concentration (ng/μl) Purity Ratios: A 260 /A 280 A 260 /A 230 Promega Corporation 29

30 Total RNA Quantification and Quality Assessment UV Absorbance Accurately measuring small RNA amounts Measurement is quantitative Affected by contaminants Standard spectrophotometer 100µL 1mL sample Sample typically diluted Nanodrop 1-2µL sample required Sensitive down to 2ng/µL Promega Corporation 30

31 Total RNA Quantification and Quality Assessment UV Absorbance Assessing RNA integrity No assessment of RNA integrity Digested RNA has similar absorbance Promega Corporation 31

32 Total RNA Quantification and Quality Assessment UV Absorbance Determining RNA Purity - Chemical carryover - DNA Contamination Pure RNA: A 260 /A 280 = 1.80 A 260 /A 230 = 2.19 Purity ratios affected by ph A 260 / A 280 : Low ratio indicates contamination A 260 / A 230 : Low ratio indicated contamination Does not indicate alcohol carry-over Genomic DNA absorbs similarly RNA % GTC A 260 /A 280 = 1.87 A 260 /A 230 = 1.16 Promega Corporation 32

33 Total RNA Quantification and Quality Assessment UV Absorbance Determining RNA Purity - Chemical carryover - DNA Contamination Pure RNA: A 260 /A 280 = 1.80 A 260 /A 230 = 2.19 Purity ratios affected by ph A 260 / A 280 : Low ratio indicates contamination A 260 / A 230 : Low ratio indicates contamination Does not indicate alcohol carry-over Genomic DNA absorbs similarly RNA + 5% EtOH A 260 /A 280 = 1.79 A 260 /A 230 = 2.19 Promega Corporation 33

34 Total RNA Quantification and Quality Assessment Gel Electrophoresis Agilent 2100 Bioanalyzer Accurately measuring small RNA amounts UV Absorbance Spectrophotometer NanoDrop /NanoVue Assessing RNA integrity Fluorescent Dye-based RT-qPCR Determining RNA Purity - Chemical carryover - DNA Contamination Promega Corporation 34

35 Fluorescent Dye-based Quantification Lower Background for Increased Sensitivity Accurately measuring small RNA amounts Dye Fluorescence proportional to amount of RNA Quantification versus an RNA standard or standard curve More sensitive than absorbance Typically requires little sample ( 1µL) Compatible with 96-well plates Excite 490nm Incubate at room temp in dark 540nm emission 490nm X Unbound dye Promega Corporation 35

36 Fluorescence-based Quantification More Sensitive than UV Absorbance QuantiFluor RNA Dye System: 100pg/µL 500ng/µL Sensitivity 20X more sensitive than absorbance Quantus + QuantiFluor RNA Dye NanoDrop * Based on using 1µl of sample per quantitation assay Promega Corporation 36

37 Total RNA Quantification and Quality Assessment Fluorescent Dye-based Quantification Assessing RNA integrity Dye No assessment of RNA integrity Dyes typically do not bind free nucleotides Incubate at room temp in dark 540nm emitted X 490nm excited 490nm Unbound dye Promega Corporation 37

38 Total RNA Quantification and Quality Assessment Fluorescent Dye-based Quantification Determining RNA Purity - Chemical carryover - DNA Contamination Dye No assessment of RNA purity Incubate at room temp in dark 540nm emitted X 490nm excited 490nm Unbound dye Promega Corporation 38

39 Total RNA Quantification and Quality Assessment Method Sensitivity Sample required Quantification Integrity Contamination detected Cost/Ease of use Gel electrophoresis Poor Qualitative Qualitative DNA Cheap, denaturing gels Agilent 2100 Bioanalyzer >50 pg/µl 1µL Yes RIN None Relatively expensive UV - spectrophotometer 100µL 1mL Diluted Yes* No Phenol, EDTA, protein, GTC Cheap, requires dilution UV - Nanodrop >2 ng/µl 1-2µL Yes* No Phenol, EDTA, protein, GTC Cheap, fast Fluorescent Dyebased *Affected by contaminants and gdna Promega Corporation 39 >20 pg/µl 1µL Yes No None Moderate cost, easy

40 Total RNA Quantification and Quality Assessment Gel Electrophoresis Agilent 2100 Bioanalyzer Accurately measuring small RNA amounts UV Absorbance Spectrophotometer NanoDrop /NanoVue Assessing RNA integrity Fluorescent Dye-based RT-qPCR Determining RNA Purity - Chemical carryover - DNA Contamination Promega Corporation 40

41 RNA Analysis Workflow Northern Blot End - point PCR Purify Quantify qpcr Protect Microarray Reverse Transcribe Next - Gen Sequencing Promega Corporation 41

42 Reverse Transcription RNA-directed DNA polymerase RNase H activity but no proof-reading Mg 2+ or Mn 2+ as cofactor Can inhibit Taq polymerase Avian Myoblastosis Virus (AMV) Moloney Murine Leukemia Virus (MMLV) Two subunits (63kDa, 95kDa) Requires 6-10mM Mg 2+ or Mn 2+ Less sensitive to 2 structure More processive Optimal activity at 42 C - 48 C Relatively high RNase H activity Used for transcripts < 5kb One subunit (75kDa) Lower RNase H activity Used for longer transcripts (>5kb) Optimal activity at 37 C MMLV H - point mutant More thermostable ( 55 C) Ideal for difficult templates >5kb Promega Corporation 42

43 Reverse Transcription Two-step RT-qPCR Making a pool of cdna Two Step RT-qPCR Reverse Transcription Heat denature RNA w/ primers Add RT, Buffer, dntps & RNasin Anneal & extend Heat inactivate RT PCR or qpcr Gene-specific primers Target mrna 5 AAA n Oligo dt Primed cdna 5 AAA n PCR Primers Random Primed cdna 5 AAA n Promega Corporation 43

44 Reverse Transcription One-step RT-qPCR Amplification of a single target One Step RT-qPCR RT & qpcr Set up as for qpcr Add RT and RNasin Use gene-specific primers Cycling considerations Perform RT first Inactivate RT/ Activate Taq Standard qpcr cycling Benefits Uses less sample Replicates over both steps Quant & QC for FFPE RNA Promega Corporation 44

45 Reverse Transcription The RT System You Choose Makes a Difference GoScript Reverse Transcriptase M-MLV Reverse Transcriptase Proprietary & optimized buffers Solutions for both stand alone RT and 1-step RT-qPCR Key Features: Efficiently transcribes long mrnas Performs better in the presence of inhibitors such as ethanol 940bp amplicon with primers at 5 end O = Oligo dt primers R = Random primers Promega Corporation 45

46 Reverse Transcription with GoScript RT Improved RT Through RNA Secondary Structure Improved RT Through RNA Secondary Structure RT GoTaq qpcr Master Mix Promega Corporation 46

47 RNA Analysis Workflow Northern Blot End - point PCR Purify Quantify qpcr Protect Microarray Reverse Transcribe Next - Gen Sequencing Promega Corporation 47

48 Promega s key products for RNA workflow ReliaPrep RNA Cell Miniprep System ReliaPrep RNA Tissue Miniprep System ReliaPrep FFPE Total RNA Miniprep System Maxwell RSC simplyrna kits Maxwell 16 System RNA Purification kits RNasin Ribonuclease Inhibitor Recombinant RNasin Ribonuclease Inhibitor RNasin Plus RNase Inhibitor QuantiFluor RNA System Quantus Fluorometer RNA Markers Diamond Nucleic Acid Dye GoScript Reverse Transcriptase AMV Reverse Transcriptase M-MLV Reverse Transcriptase, RNase H- Point Mutant Go Taq 2-Step RTqPCR System Go Taq 1-Step RTqPCR System Maxwell 16 LEV FFPE Purification kit Promega Corporation 48

49 Technical Services Scientists Ready to Help Promega Corporation 49

50 Questions Welcome Promega Corporation 50