Isolating Total RNA? Scott Tighe 802-656-2557 HSRF 305

RNA Structure Major Types mRNA-transcription Different from DNA rRNA- 5s,5.8s,16s, 18s,23s,26s 28s tRNA-Involved in PS Other ncRNA- miRNA, siRNA snRNA snoRNA SmY scaRNA gRNA RNase P RNase MRP Different from DNA has 2’OH Group!

Degradation of RNA-Two Major Catagories Catalytic/ Enzymatic RNases -Catalytic His 12 and His 119, produce a 2’-3’ cyclic phosphate intermediate similar to chemical catalysis Ribozymes-RNase P Chemical Catalysis by Acid, Base, Divalent Ion 2’ oxygen attacks the adjacent phosphate 2’ OH transesterification Yingfu Li, and Ronald R. Breaker J. Am. Chem. Soc., 1999, 121 (23), 5364-5372

Why Total RNA? Not all transcripts have poly A tail- ie mitochondrial RNA assessment is more determinative rRNA subunit have decrete peaks when running a gel or Bioanalyzer Recovery of special RNA’s such as miRNA, NC RNA, nuclear RNA ect mRNA recovery kits also recover rRNA anyway Total RNA mRNA

General RNA Handling Reagents and Equipment-Considerations: All reagents MUST be RNase-free Use gloves that are periodically treated with RNase Zap Perform all work in a hood Biosafety Laminar flow PCR hood DO NOT use a fume hood Use aerosol resistant pipet tips ONLY Prepare all surfaces and pipets by treating with RNAse Zap All utensils [scissors, scalpels, tweezers] should be scrubbed clean, sprayed with RNase Zap, soaked in ETOH and flame sterilized before a surgery

General RNA Handling Prepare daily aliquots of RNase-free water. I aliquot 10-20 tubes each week and discard half way through the day. Diethylpyrocarbonate [DEPC]-treated water is NOT an inhibitor for RNases, but rather DEPC is a chemical added to water to eliminate RNases. After autoclaving [or when purchased], no DEPC resides in the water. When opening and closing tubes, be careful not to bump the inner rim of your tubes. Use a RNase Inhibitor if allowable by downstream reactions RiboLock Superase others

Why an RNase Inhibitor? RNase Test Time-0 With RI No RI

RNases are almost everywhere Results from an RNase test system Surface treated RNase A followed by Decontamination Several RNase Inhibitors

RNA Extraction Systems

Silica Column-based Most use 4M guanidine isothiocyanate [GCN] chaotropic salt system that denatures RNases without the use of phenol RNA is precipitated with ethanol and bound to silica [Si-O-H], washed, and eluted with water. PROS CONS Easy to perform Lipids will interfere and inhibit Compatible with Shedder column Will not isolate MiRNA No precipitation rxn Will not isolate RNA <200bp Very clean RNA Lower yield than Trizol Compatible with FastPrep Salts may be left behind DNase on the column RLT Poor storage integrity at -80 Abrasives can end up in final sample heavy DNA contamination problems

RNA Isolation and Purification Systems-[Column-based] RNeasy Micro kit [74004] Small elution volumes 10-15ul for 10ug Good for FACS samples, LCM, or limited cell On-column DNase treatment RNeasy Mini Kit [74104] Standard Elution volume of 30-50ul for 100ug General use column On column DNase treatment Lipid or Fiber kits too RNeasy Midi and Maxi Kit Large elution volumes 150-800ul for 1mg of RNA USB Corp Prep-Easy Kit Invitrogen Pure Link micro Ambion RNAqueous Zymo Corp-Not recommended

Trizol-based Reagents Phenol-Guanidine reagent TriZol Tri-Reagent TriSure Purezol QiaZol Three types Trizol – Standard-1:9 ratio must be maintained [10% sample] Trizol LS -Concentrated-1:3 ratio must be maintained [33% sample] Trizol BD-designed specifically for blood Remove extracellular biomolecules with chloroform or bromochrolopropane Requires a precipitation reaction Use Axygen MCT175C ultra clear tubes better pelleting easier to see May add Pellet Paint, GlycoBlue to precipitation reaction for low [ ] of RNA

Use both a Trizol (guanidine-phenol) reagent and silica column Hybrid Systems Use both a Trizol (guanidine-phenol) reagent and silica column Trizol Plus system [Invitrogen] Qiagen RNeasy Lipid Tissue Mini Kit Bio-Rad Aurum Total RNA Fatty and Fibrous Tissue Kit Home Made: Perform the standard Trizol Transfer Aqueous phase to new tube Add 1.5x volume of 100% ETOH Apply to column Follow standard column proceudures Sometimes leaves a Trizol residue on low recovery samples

Considerations on which system to use TriZol Applications Tissues high in lipid and other cellular biomolecules will require Trizol because of potential interferences of the Si-OH Trizol has a higher recovery of RNA then RNeasy columns but not useful for very small amounts of RNA [+/-] Must be cleaned and DNase-treated using column Use of Bromochloropropane instead of chloroform for higher purity. 260/280 ratios are often 1.6-1.8 with chloroform and 1.8-2.0 for BCP. loss of the 28S subunit for solid tissue Advantageous when grinding matrix is hard to remove- it spins out Can recover RNA and DNA

Considerations on which system to use Silica Column-based Applications [RNeasy] General use and rapid No precipitation reaction need Optimized columns for very small concentrations On-column one step DNase Treatment No Phenols or organic solvents Grinding matrix can end up in the sample Recoveries of approximately 60% of Trizol (we’ve seen) DNase-treatment will generally reduce yield by 30% or so

Considerations on which system to use Silica Column-based Applications [RNeasy]…continued Use either centrifuge or vacuum manifold-Multiple loadings Heat elution water to 60C will increase yield Do not spin with column open 260/280 ratios are often above 2.00 If using MinElute or MicroElute columns-be aware of the o-ring-it catches and retains liquid that can get into your final sample

Extracting RNA

Extracting RNA from Tissue RNA integrity is a function of tissue type and handling! Column or Trizol or ….Trizol follow by a column clean-up Know your tissue characteristics before starting High RNase content tissues Spleen Pancrease Intestine Thymus Connective tissues, collagen, protein, glycogen, lipids can interfere with silica column Brain Liver Heart Muscle Adipose

Extracting RNA from Tissue Use fresh tissue when possible Use a hood Use only utensils that are disposable RNase-free or treated with RNase Zap,ETOH, and flame sterilized!!! Take special precautions when working with challenging tissues such as glazing tissue with RNase inhibitor Place directly into extraction reagent and extract immediate [i.e. homogenize] GCN TriZol DO NOT overload the extraction reagent Interferance of DNA, lipids or polysaccharide can inhibit silica reaction

Extracting RNA from Tissue- Types of homogenization Traditional mortor and pestle with LN2 Liquid Nitrogen is not always RNase-free Difficult to make sterile and RNase-free Poly-tron with new tips or RNase-free blades Not optimized for very small quantities Sometimes difficult to make RNase-free Mini motorized pestle With or without abrasive All RNase-free disposable Impactor [bio-pulverizer] French Press Biomasher columns

FastPrep System - Mini-bead beater Automated, fast, RNase-free Uses screw cap 2ml tubes, 15ml and 50ml and optional abrasive for homogenizing tough tissue Excellent for bacterial extractions In Room HSRF 305-Sign up

Extracting RNA from adherent cells Directly from plate or dish Similar for both Trizol and RNeasy system Do not trypsinize Remove growth media and washing with PBS (with or w/o RI) Add enough extraction reagent fro the number of cells Vortex plate directly Follow standard operating procedure

Extracting RNA from other Sources Suspended mammalian cells Centrifuge to a pellet Wash with PBS(RI) to remove serum and media Add extraction reagent and vortex Follow SOP Viability is key to recovering intact RNA- Trypan or Eosin Bacteria and yeast RNA’s are best recovered during early log phase! Heating of Trizol or RLT buffer to 50C May require FastPrep with abrasive Cell wall digestion (Lyticase, Prok, Lysozyme)

Quality Control of RNA

Consider running an RNase-free gel Quality Control of RNA Consider running an RNase-free gel Look for gDNA Look for rRNA bands We use the E-gel and FlashGels

Can not distinguish DNA from RNA Measure your RNA on the Nanodrop and Bioanalyzer Can not distinguish DNA from RNA Can not distinguish degraded RNA from “good” RNA Good RNA RNA prep with mostly gDNA from Neutrophils

Good vs Degraded RNA Thing are not always as they appear- These look great on the nanodrop… but…. are they?

Expected Yield Expected yield is very important!!! Just getting “enough” RNA is not always ok Actively growing mammalian cells contain 1-10 pg/cell Calculate your expected RNA recovery If you are way below your expected yield than…. Selectively recovered RNA from weak or apoptotic cells Selectively recovered RNA from G0 or G2M Will significantly impact gene expression data

Problematic Nanodrop Traces Sample is NOT 183 ng/ul Actually 38ng/ul as per Qubit Spectrofluoromater 272 nm peak is skewing data 260 272 RNA How will this affect downstream processes such as RT-qPCR if one assumes equal RNA input to a cDNA reaction? TRZ

Nanodrop Spectra EDTA GUAN-HCL RLT Trizol GUAN-ISOThio Tween 20

Nanodrop Spectra Glycogen Amm. ace Sod. ace PCI MES Salt Alginate

Nanodrop Spectra Residual Trizol ETOH RNase Inhibitor Tris

Round table discussion

FACS and LCM

FACS sorted cells and RNA 1] FACS must be RNase-free by bleach and other treatments 2] Bacterial contamination in sheath tanks and dip tubes can cause RNases 3] Hold back cells –check viability- extract RNA as a pre-sort control 4] Add Superase or RiboLock to sort tube and pre-sort tube containing cells 5] Use RNase-free tubes to sort

6] Sort into an exact volume of ice cold extraction reagent Trizol LS RLT buffer [RNeasy Micro] Media or buffer with or without Superase 7] After sorting measure the total volume and add the necessary volume of reagent to obtain the correct ratio of extraction reagent to sort liquid 8] Consider acceptable sort volumes 9] Extract immediately-do not store cells in extraction buffer at -80 10] DNase-treatment causes RNA losses using RNeasy

LCM and RNA Test tissue section before starting by aseptically removing and extracting a small section. DO NOT LET FROZEN TISSUE THAW! FFPE tissues often yield no usable RNA and testing its condition before the start is a good idea Know what level of degradation your downstream application can tolerate Fresh frozen tissues perform well Use RNase Zap-ETOH treated microtome with new blade during sectioning Use RNase –free slides, tweezers, materials

LCM Scape a small section from the prepared slide and extract as a control Prepare all staining and dehydration reagents from RNase-free reagents and DEPC water Collect one LCM cap from large area of cells as a control-non-specific cells Collect target cells and transfer cap to tube containing extraction agent-vortex Extract immediately We have had good luck with RNeasy micro and Pico Pure kits Omit DNase step to increase recovery when allowable Extract several caps into one extract buffer or several extract buffers and combine to increase yield

Thank you for your attention!