1. Isolating Total RNA?
Scott Tighe
HSRF 305

2. 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!

3. 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),

4. 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

5. 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

6. General RNA Handling
Prepare daily aliquots of RNase-free water. I aliquot 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

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

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

9. RNA Extraction Systems

10. 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

11. 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 ul for 1mg of RNA
USB Corp Prep-Easy Kit
Invitrogen Pure Link micro
Ambion RNAqueous
Zymo Corp-Not recommended

12. 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

13. 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

14. 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 with chloroform and 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

15. 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

16. 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

17. Extracting RNA

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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)

24. Quality Control of RNA

25. 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

26. 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

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

28. 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

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

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

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

32. Nanodrop Spectra
Residual Trizol
ETOH
RNase Inhibitor
Tris

33. Round table discussion

34. FACS and LCM

35. 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

36. 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

37. 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

38. 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

39. Thank you for your attention!