RNA ISOLATION and PURIFICATION

SELECTING A PURIFICATION STRATEGY Do Your Experiments Require Total RNA or mRNA? the researcher must be has to make when detecting or quantitating RNA is whether to isolate total RNA or poly(A)-selected RNA(mRNA).

This choosing is complicated by: the bewildering array of RNA isolation kits available in the marketplace.
the downstream application influences this choice. total RNA might suffice for most applications. the starting material for applications ranging from the detection of an mRNA species by Northern hybridization to quantitation of a message by RT_PCR.

You remember that: mRNA comprises (<5% )of cellular RNA. the potential loss of a particular
message species during poly(A) purification,

the difficulty in quantitating small amounts of purified poly(A) RNA.

How do resolve this problem?

If data generated with total RNA do not meet your expectations, using poly(A) RNA instead might provide the( sensitivity )and specificity that your application requires.

You must be detect The pros and cons of each application.

Two situations where using poly(A) RNA is essential are: 1.cDNA library construction. 2.preparation of labeled cDNA for hybridization to gene arrays.

Northern blotting or Northern hybridization

is a technique for detection of specific RNA sequences.
Northern blotting was developed by James Alwine and George Stark at Stanford University and was named such by analogy to Southern blotting.

Northern blotting involves the following steps:

1) Total cellular RNA or mRNA is size-separated by denaturing agarose gel electrophoresis, 2) 2) The separated RNA is transferred onto a nylon membrane;

3) 3) The RNA is then detected with isotopic or non-isotopic labeled complementary DNA or RNA probe

Northern Hybridizations
Northern analysis is the only technique available that can determine the molecular weight of an mRNA species.

Total RNA is most commonly used in this assay.

Since only very small amounts of poly(A) RNA are present, make sure that it is feasible and practical to obtain enough starting cells or tissue. Example .if you use 30micro gram of poly(A) RNA in a Northern ,which is the amount found in approximately 1mg of total RNA.

Will it be practical and feasible for you to sacrifice the cells or tissue required to get this much RNA.?
If not, use as much poly(A) RNA as is practical.

If using poly(A) RNA in Northern hybridizations.

is the absence of the ribosomal RNA, which are ordinarily used to gauge the quality and relative quantity of the RNA samples In your research.

there are other strategies besides switching to poly(A) RNA that can be used to increase the sensitivity of Northern hybridizations.
You could alter the hybridization conditions of the DNA probe you could switch to using RNA probes in the hybridization, RNA probe are( 3 to 5)fold more sensitive than DNA probes in typical hybridization buffer.

Dramatic differences in the sensitivity of Northern blots can also be seen from using different hybridization buffers.

Nuclease protection assay .is a technique used to identify individual RNA molecules in a heterogeneous RNA sample extracted from cells. The technique can identify one or more RNA molecules of known sequence even at low total concentration. RT-PCR (reverse transcription-polymerase chain reaction) is a sensitive method for the detection of mRNA expression levels. Traditionally RT-PCR involves two steps: 1.the RT reaction . 2.PCR amplification. RNA is first reverse transcribed into cDNA using a reverse transcriptase . the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes.

If you are not interested in determining the size of the target, you can use more sensitive technique such as: 1. nuclease protection assay. 2. RT-PCR.

Nuclease protection assays, which are 5- to 10-fold more sensitive than traditional membrane hybridizations.
Can accommodate 80 to 100micro gram of nucleic acid in a single experiment. RT-PCR can detect extremely rare messages, for example, 400 copies of a message in a 1micro gram.

RT-PCR is currently the most sensitive of the RNA analysis techniques. it is enable to detection and quantitantion of the rarest of targets.

IF you want quantitative approaches have become increasingly reliable with introduction of internal standards you must use competitive PCR strategies.
For quantifying mRNA, internal standard RNAs are added in a defined quantity to the RNA sample prior to the RT reaction. The resulting standard cDNA is co amplified with the same primers as the endogenous target sequence. Its PCR product is approximately 50 nucleotides smaller. This method allows measurement of small differences (as low as factor 2) in mRNA amount between RNA samples.

Dot slot blot

RNA samples are directly applied to a membrane manually or under vacuum through a filtration manifold. Hybridization of probe to serial dilutions of sample can quickly generate quantitative data about the expression level of a target. Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from anagarose gel to a membrane support (northern blotting). unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis

Total RNA or poly(A) RNA can be used in this assay since the RNA is not sizefractionated on an agarose gel.

using total RNA in dot/slot blots is that signal of interest cannot be distinguished from cross-hybridization to rRNA.

Switching to poly(A) RNA as the target source might alleviate this problem. Hybridization to Gene Arrays and Reverse Dot Blots Gene arrays consist of cDNA clones as a form of oligonucleotides or PCR products. oligo spotted at high density on a nylon membrane

glass slide, other solid.

PCR product by: hybridizing labeled cDNA probes reverse transcribed from mRNA. the expression of potentially hundreds of genes can be simultaneously analyzed.

Procedure requires that the labeled cDNA be present in excess of the target spotted on the array.

This is difficult to achieve unless poly(A) RNA is used as template in the labeling reaction.

Ribonuclease Protection Assays

total RNA or poly(A) RNA can be used as starting material in this assay. total RNA usually affords enough sensitivity to detect even rare message. maximum amount ( 80 to 100micro gram). If the gene is expressed at extremely low levels, requiring weeklong exposure times for detection. if you want switch to poly(A) RNA might prove beneficial and may justify the added cost. This technique is very sensitive,because of do require laborious gel purification of the full-length probe to avoid getting confusing results.

RT-PCR RT-PCR is the most sensitive method for detecting and quantitating mRNA.
very low-abundance messages can be detected with this technique. Total RNA is routinely used as the template for RT-PCR.

some cloning situations and rare messages require the use of poly(A) RNA
2 different schools of thought concerning RT-PCR. First ,it advisable to treat the sample RNA with(DNase I )(no purification). method produces RNA completely free of contaminating genomic DNA. very small amounts of genomic DNA contamination can cause false positives . A second school of thought avoidance of DNase I.

cDNA Library Synthesis

high-quality mRNA that is essentially free of ribosomal RNA is required for constructing cDNA libraries. Unacceptably high backgrounds of ribosomal RNA clones. would be produced if total RNA were reverse transcribed to prepare cDNA.

cDNA is a DNA copy synthesized from mRNA. T RNA-dependent DNA polymerase isolated from a retrovirus (AMV or MMLV). This is provided by the poly(A) tail found at the 3' end of most eukaryotic mRNAs . which a short complementary synthetic oligonucleotide (oligo dT primer) is hybridized . (polyT -polyA hybrid).

Is It Possible to Predict the Total RNA Yield from a Certain Mass of Tissue or Number of Cells? Sections are based on experimentation at Ambion.
Ambion laboratory technique using a variety of samples and different purification products. YIEYLD can vary widely based on the type of tissue or cells used for the isolation.

rapid and complete tissue disruption, and homogenizing at subfreezing temperatures cannot be overemphasized.
because yields from very small amounts of starting material are subject to the law of diminishing returns.

choose more starting material rather than less. Samples can be pooled together, if possible, to maximize yields.

For example, if your sample is 5 mg of tissue or 2.5 X 106 cells You can yields about 10micro g of total RNA,

8microg rRNA,
0.3microg mRNA , 1.7micro g tRNA , and other RNA . 1 g of tissue or 5X108 cells.

yields about 2mg of total RNA 1.6mg rRNA 60mg mRNA 333mg tRNA and other RNA.

Protein contamination.
Pure RNA has an A260:A280 absorbance ratio of 2.0. in RNase protection assays.

Northern analysis.
vitro translation experiments. RT-PCR assays used total RNA with A260:280 ratios ranging from 1.4 to 1.8 . If protein contamination occure is causes to change in these ranges. Resolving problem is by: using additional organic extractions with anequal volume of (phenol/chloroform/isoamyl alcohol) (25 : 24: 1mixture) may remove the contaminant.

Residual phenol can cause lower the A260:A280 ratio,

inhibit downstream enzymatic reactions. Chloroform/isoamyl alcohol (24 : 1) extraction will remove residual phenol.

Chloroform causes proteins to become denatured and become soluble in the organic phase or interphase, while nucleic acids remain in the aqueous phase. Phenol dissolves proteins and lipids leaving water soluble matter.
liver and kidney tissues, produce RNA consistently at a lower absorbancy ratio than the other absorbancy ratio for these tisuue is rarely above 1.7

Physically Intact?
The integrity of your RNA is best determined by electrophoresis on a formaldehyde agarose gel under denaturing conditions. samples can be visualized by adding 10microgm/ml of EtBr to the sample before loading on the gel. The most sensitive test of RNA integrity is Northern analysis using a high molecular weight probe expressed at low level in the analyzed tissue.

Northern analysis is not tolerant of partially degraded RNA. the quality of the data is severely compromised. IF a single cleavage in20% of the target molecules will decrease the signal on a Northern blot by 20%.

Nuclease protection assays and RT-PCR are tolerant.

Which Total RNA Isolation Technique Is Most Appropriate for Your Research?
There are three basic methods of isolating total RNA from cells and tissue samples.

Chaotropic agent use to open the cells and inactivate RNases.

The lysate is then processed in one of several ways to purify the RNA away from protein, genomic DNA, and other cellular components.

These 3 methods include: Guanidium-Cesium Chloride Method

1. Harvest 25 g of tissue, freeze in liquid nitrogen (N2) and grind to a powder under liquid N2 with a mortar and pestle. 2. Resuspend powder in 150 mL of buffer containing 4.5 M guanidinium thiocyanate /50 mM EDTA (pH 8.0), 25 mM sodium citrate (pH 7.0), 0.1 M

2-mercaptoethanol, 2% lauroylsarcosine.

3. Homogenize suspension on amedium setting for 3-5 minutes at 4 C.

4. Remove insoluble material bycentrifuging the suspension in aFiberlite F1314x50cy carbon fiber rotor at 8,000 xg for 10 minutes at 4 C.

5. Add CsCl to the supernatant at concentration of 0.2 g/ml

6. Layer the sample solution over10 mL of 5.7 M CsCl containing 50 mM EDTA (pH 7.3). If using apolyallomer tube, ensure the tube is completely full and the 5.7 M CsClcushion should occupy one-third of the total tube volume.ifnecessarytop off tube with 0.2 g/ml CsCl buffered solution.

7. Centrifuge sample solution inFiberlite F50L-8x39 rotor at 50,000 rpm (~266,000 xg) for 5 hours at 20C.
8. After centrifugation, remove RNA pellet and dissolve in 2.0 ml of 10 mM Tris-HCl (pH 7.4), 2 mM EDTA. 9. To further purify, extract RNA3 times with 2 volumes of phenol/chloroform (1:1) followed by1 extraction with chloroform (Total RNA yield = 3.2 mg).

10. Isolate poly(A)+ RNA by 2 cycles of oligo(dT)-cellulose chromatography(5) (Final poly(A)+yield = 42 g).

disadvantage
RNA is more dense than DNA and most proteins, it pellets at the bottom of the tube after 12 to 24 hours of centrifugation at 32,000rpm. Small RNAs (e.g., 5S RNA and tRNAs)can not be prepared by this method. time-consuming, laborious, and required overnight centrifugation. The number and size of samples that could be processed simultaneously were limited by the number of spaces in the rotor.

Single- and Multiple Step Guanidium Acid-Phenol Method The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation.

Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase.
Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples.

why guanidium-acid phenol procedure has largely replaced the cesium cushion method? Because of: because RNA can be isolated from a large number of samples in two to four hours.
the technique can be easily scaled up or down to process different sample sizes. is based on the propensity of RNA molecules to remain dissolved in the aqueous phase in a solution containing 4M guanidium thiocyanate.ph4 DNA remain in the organic phase. proteins and other cellular macromolecules are retained at the interphase.

Problem during the phenol/chloroform extraction step The mixture must be spun with sufficient force to ensure adequate separation of the organic and aqueous layers. depend on the rotor.

The interface between the aqueous and organic layers is another potential source of genomic contamination.

Avoid the white interface (cream colored or brownish) between the two layers; leave some of the aqueous layer with the organic layer.
Residual salt from the precipitation step, appearing as a huge white pellet, can interfere with subsequent reactions. Excess salt can be removed by washing the RNA pellet with 70% EtOH (ACS grade).

Remove the ethanol carefully, as the pellets may not adhere tightly to the tubes ethanol removed by aspiration with a drawn out Pasteur pipet after re spun.

Non-Phenol-Based Methods (phenol-free methods).

Properities: Very fast, clean RNA, can process large sample numbers.
Free from the handling and disposal of phenol, a very hazardous chemical. yields total RNA of the same quality as the phenol-based methods.

based on the ability of glass fiber filters to bind nucleic acids in the presence of chaotropic salts like guanidium,
requiring no organic extractions. processing large sample numbers is fast and easy. the quickest methods for RNA isolation completed in less than one hour

Problems.
DNA contamination can be higher with this method than with phenol-based methods. The primary problem is clogging of the glass fiber filter by thick lysates. This can be prevented by using a larger volume of lysis buffer initially. Second problem is to minimize the viscosity of the lysate by sonication.(on ice, avoid power settings that generate frothing) . If your sample is enriched by high in saccharides or fatty acids an initial clarifying spin or extraction with an equal volume of chloroform can prevent filter-clogging problems.

frothing amass of bubbles in or on a liquid foam.

You can solve by clarifying spin is 8 minutes at(7650 Xg.). if you failed in this solving the lysate can be divided into micro centrifuge tubes and centrifuged at maximum speed for 5 to 10 minutes. if your sample is rich in glycogen such as liver, or plants containing high molecular weight carbohydrates. Avoid initial clarifying spins on tissues. If you generate a clogged filter, remove the remainder of the lysate using a pipettor. place it on top of a fresh filter, and continue with the isolation protocol using both filters.

steps:
1.the cells are first lysed in a guanidium-based buffer. 2.The lysate is then diluted with an organic solvent such as ethanol or Isopropanol.

3.applied to a glass fiber filter or resin.

4.DNA and proteins are washed off, and the RNA is eluted at the end in an aqueous buffer.

What Protocol Modifications Should Be Used for RNA Isolation from Difficult Tissues?
Protocol modification necessary to eliminate specific contaminants, or tissue treatments prior to the RNA isolation protocol.like Fibrous tissues, tissue rich in protein, DNA and RNases, Type of sample cells and tissues. Fibrous Tissue. Like heart and muscle Due to low cell density and the poly nucleate nature of muscle tissue You do it pulverizing the frozen tissue into a powder while keeping the tissue completely frozen (use a chilled mortar and pestle) is the key to isolating intact total RNA.

Lipid and PolysaccharideRich Tissue Such as Plant and brain tissues. difficult to get clean separation of the RNA

If you use phenol-based methods to isolate total RNA, white flocculent material present through out the aqueous phase.
This flocculate will not accumulate at the interface. Chloroform: isoamyl alcohol (24 : 1) extraction of the lysate is probably the best way to partition the lipids away from the RNA. To minimize loss, back-extract the organic phase, and clean up the recovered aqueous RNA by extraction with phenol : chloroform : isoamyl alcohol (25 : 24:1).

When your sample is plant tissue using a non-phenolbased method, polyvinylpyrrolidone-40 (PVP-40) can be added to the lysate to absorb polysaccharide and polyphenolic contaminants . When the lysate is centrifuged to remove cell debris, these contaminants will be pelleted with the PVP Centrifugation on cesium tri fluoroacetate has also been shown to separate carbohydrate complexes from RNA

The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA .

Nucleic Acid and Nuclease-Rich Tissue Spleen and thymus Best homogenization is the key to isolating high-quality RNA from these tissues. samples should be completely pulverized on dry ice, under liquid nitrogen, to facilitate rapid homogenization in the lysis solution, and cause inhibits nucleases. If your sample is Cancerous cells contain high amounts of DNA and RNA. These cells are unusually viscous.causing poor separation of the organic and , aqueous phases and potentially clogging RNA binding filters.

Increasing the ratio of sample mass : volume of lysis buffer can help alleviate this problem in filter-based isolations.

How can you resolve this problem?

Increasing the ratio of sample mass : volume of lysis buffer can help alleviate this problem in filter-based isolations. Multiple acidphenol extractions can be done to ensure that most of the DNA is partitioned into the organic phase during acid phenol based isolation procedures. If a lysate of your sample is viscous by attempting to pipet the solution and observing whether it sticks to the pipette tip. The DNA in the lysate can alternatively sheared. by vigorous and repeated aspiration through a small gauge needle (18 gauge) or by sonication (10 second sonication at 1/3 maximum power on ice, or until the viscosity is reduced.

Hard Tissue bone and tree bark. disrupted using a PolytronTM or any other commonly available homogenizer. Heavy-duty tissue grinders that pulverize the material using mechanical force are needed. SPEX Certiprep. Tissue-grinding mills that chill samples to liquid nitrogen temperatures and pulverize them by shuttling a steel piston back and forth inside a stationary grinding vial.

Bacterial and yeast cells

quite refractory to isolating good-quality RNA due to the difficulty of lysing them
bacteria is the short half-life of most bacterial messages.

Lysis can be facilitated by re suspending cell pellets in TE and treating with lysozyme

TE use to solubilize the RNA and protect from the degredation.

They can introduce RNases. use the highest quality enzymes to reduce the of introducing contaminants. You can obtainYield and quality by use phenol-based extraction protocols can be improved by conducting the organic extractions at high temperatures.

Yeast cells is accomplished by vigorous vortexing in the presence of 0.4 to 0.5 mm glass beads. Yeast cells can be treated by zymolase, lyticase, and glucolase to lysis.

Poly(A) RNA Purification Strategies.

purify poly(A) RNA directly from the starting material via by one step strategy.
first isolates total RNA, then purifies poly(A) RNA from that by 2 steps strategy. These 2 different steps are difference in number, size of sample and yield produce.

Making and using mRNA (2)

One-step strategies involve fewer manipulations to recover poly(A) RNA

Two additional washing steps multiplied by 20 samples consume significant time and materials, and arguably, faster.

purification strategies decrease the chance of degradation

The true speed of a technique is determined by the total manipulations in a procedure. one step procedures is used in hybridization of gene array. Percentage of poly(A) RNA is similar between one- and two-step strategies. But when experimental sample is limited, a one-step procedure is the more practical procedure.

one-step products are usually geared to purify small (15microg) or large (25microg) quantities of poly(A) RNA. If you require more poly(A) RNA, a two-step procedure is usually more cost effective. RNase-Free Technique RNase contamination is prevalent, You must be prepare a solutions to dissolve problem should be prepared in disposable. RNase-free plastic ware .

RNase-free glassware prepared in the lab.

Glassware: by baking RNaseat 180C for 8 hours to overnight.

Treating with a commercial RNase decontaminating solution.

Also you can solve Rnase problem by: RNase can be removed by filling containers with 0.1% DEPC. incubating at 37C for 2 hours. Rinsing with sterile water . Heating to100C for 15 minutes. Autoclaving for 15 minutes to eliminate RNase. Electrophoresis apparatus used for RNA analysis can be made: RNase-free by filling with a 3% hydrogen peroxide solution.

Incubation period 10 minutes at room temperature.

Rinsing with DEPC-treated water.

DEPC inactivates RNases by carboxyethylation of specific amino acid side chains. DEPC is a suspected carcinogen.be careful with handling it.

Diethyl pyro carbonate.?how it act ?

this inactivates the residual DEPC by hydrolysis causes to Releases CO2

EtOH as by-products

The EtOH by-product can combine with trace carboxylic acid contaminates in the vessel to form volatile esters. increasing the concentration of DEPC to 1% can inhibit more RNase but can also inhibit certain enzymatic reactions,is usually not better.

DEPC-Treated Water It should be noted that many reagents commonly used in RNA studies contain primary amines, such as Tris, MOPS,HEPES, and PBS, and cannot be DEPCtreated because the amino group sops up the DEPC. making it unavailable to inactivate Rnase. MOPS(N-morpholino)propane sulfonic acid,

HEPES (4-2-hydroxyethyl)-1-piperazineethanesulfonic PBS: phosphate buffered saline

acid )

How Do You Minimize RNA Degradation during Sample Collection and Storage?
RNase is present in all cells and tissues they must be immediately inactivated when the source organism dies. Samples should be immediately frozen in liquid nitrogen.

In other experiments homogenized tissue has been stored for at least one week at room temperature or two months at 4C without any loss of RNA in a lysis buffer .

A commercial RNase inhibitor also exists that can prevent RNA degradation within mammalian tissue, cells, and plant tissues stored above freezing temperature for long periods.

Safe Place to Pause during an RNA Purification Procedure. Ideally RNA should be purified without interruption. stop when the RNA is precipitated or is in the presence of a chaotrope.

For example, when using an organic isolation procedure, the RNA isolation can be stopped when the samples have been homogenized in a chaotrophic lysis solution.
They can be stored for a few days at -20C or -80C without degradation.

STORAGE OF PURIFIED RNA.

short-term storage a few weeks or less, store your RNA in RNase-freeTris-EDTA or 1mM EDTA at -20C in aliquots.

Long term storage. RNA should be stored in aliquots at -80C in TE,1mM EDTA, formamide, or as an ethanol/salt precipitation mixture.