RNA Extraction

Ribonucleic Acid

RNA stands for?

RNA

A type of nucleic acid with only one strand

Ribose

RNA has this type of sugar, instead of deoxyribose

Uracil

RNA uses this on its nitrogen base instead of thymine (in DNA)

RNA

Provides the link between the genetic information through protein synthesis (serve as template for protein synthesis).

rRNA, mRNA, tRNA, other molecules

Total RNA is composed of?

85%

Total RNA:
Percentage of rRNA

2%

Total RNA:
Percentage of mRNA

10-15%

Total RNA:
Percentage of tRNA and other molecules

1960, Kurland, CsCl centrifugation

History of RNA Isolation and Purification
In the year ______, ________ and colleagues performed an RNA isolation procedure using _________________ technique.

1968, Kirby, phenol, chloroform

History of RNA Isolation and Purification
In the year ______, another method was introduced by ________ using _______ and _________.

1979, Chirgwin, GTC, Beta-Mercaptoethanol, ethanol extraction, ultracentrifugation, CsCl gradient

History of RNA Isolation and Purification
In the year ______, _________ introduced a method using _____ and _________; followed by ____________ and ___________ in ________.

1987, Chomczynski, Sacchi, guanidium thiocyanate, phenol, chloroform

History of RNA Isolation and Purification
In the year ______, __________ and _________ combined all extraction steps in one single procedure using ____________, __________, and __________.

Different from the DNA working area (if possible).

Important Reminders during RNA Isolation
(1) The RNA working area should be ________________________________________.

finger RNases

Important Reminders during RNA Isolation
(2) Gloves should be worn always and changed frequently to avoid introduction of _________________.

capped, covered, dust, other particles

Important Reminders during RNA Isolation
(3) All tubes and bags should be ________ and ________ when it’s not used to avoid contamination with ___________ and _________.

pipettors, before, after, ultraviolet light, Lysol, alcohol

Important Reminders during RNA Isolation
(4) Sterilize area and other materials, such as ___________, ______ and ______ working. Use __________ or spray with _________ and _________.

purity, quality, quantity

Important Reminders during RNA Isolation
(5) One RNA isolation is finished, the work is not done yet until the ________, _________ and ________ of RNA is checked.

Microarray analysis, Northern analysis, cDNA library construction, RT-PCR

Purpose of RNA Extraction
Isolation of intact RNA is essential for many techniques used in gene expression analysis such as:
(1)
(2)
(3)
(4)

Freeze the samples in liquid nitrogen, Immediately disrupt fresh samples, Store the samples

Handling Samples Prior to RNA Extraction
(1)
(2)
(3)

acidified phenol, chloroform, proteins, lipids, DNA, alcohol precipitation

Organic Extraction: How it Works
Organic extraction is composed of ________ and _________. It removes ___________, _________ and ________ from the RNA sample. RNA is then recovered by ______________.

large, small, modify, high levels, fat, protein

Organic Extraction: Advantages

• can be used for ______ or ______ sample sizes.

• Can ________ extractions to remove ________ of _____ and ________ from samples.

Phenol, chloroform

Organic Extraction: Disadvantages
________ and ________ are hazardous.

Glass filters, other cellular components, eluted, highly purified form`

Column Purification: How it Works
_________ bind the RNA while ______________ are washed away. RNA is ________ in a _____________.`

Rapid procedure, organic solvents, alcohol precipitation

Column Purification: Advantages

• ___________

• No ___________ required.

• No _______________ needed.

scalable, organic extraction methods

Column Purification: Disadvantages
Not as _________ as ____________.

Cell lysis and disruption of cellular structures.
Separation of the RNA from cell debris.
Purification of the RNA from the DNA and proteins.
Precipitation of RNA.
Wash and re-suspension of the RNA.

RNA Isolation Steps:
The main steps of RNA isolation are as follows:
(1)
(2)
(3)
(4)
(5)

Use of liquid nitrogen, Use of glass beads, Use of commercially available kit, Use of repetitive pipetting

RNA Isolation Methods:
Several methods of RNA isolation are available depending on the type of samples and depending on the materials/reagents and instrumentation available. There will be four methods of RNA isolation to be performed:

Homogenization, Phase separation, RNA Precipitation, RNA Washing, RNA Dissolution

RNA Isolation Steps: Use of liquid nitrogen
(1)
(2)
(3)
(4)
(5)

Homogenization, Phase separation, RNA Precipitation, RNA Washing, RNA Dissolution

RNA Isolation Steps: Use of glass beads
(1)
(2)
(3)
(4)
(5)

Sample Preparation, RNA Purification, RNA Washing

RNA Isolation Steps: Use of commercially available kit
(1)
(2)
(3)

Homogenization, Phase separation, RNA Precipitation, RNA Washing, RNA Dissolution

RNA Isolation Steps: Use of repetitive pipetting
(1)
(2)
(3)
(4)
(5)

DNase Treatment of RNA Samples

Additional Step in RNA Isolation Steps

Liquid nitrogen, low temperature environment, enzymes in low temperature, degradation

_________________ was used to provide __________ environment, inhibit ____________ and prevent RNA from __________ in grinding process.

-20C, -80C, liquid nitrogen

Traditionally RNA is stored at _______, _______ or in _______ to provide protection from these degradative reactions.

snap, flash, dry ice, liquid nitrogen

Tissues that need to be stored prior to RNA isolation are “_____” or “_____” frozen on ________ or in _________ to preserve RNA integrity.

Glass beads, eppendorph tube, physically break, french press, sonification device

____________ are added to an __________ containing a sample of interest and bead beater vigorously vibrates the solution causing the glass beads to ____________ apart the cells. Other methods used for lysing cells include a ___________ and a ____________.

high quality purified RNA, sequencing, transcriptome analysis, infectious pathogen testing

The purpose of RNA Extraction is to obtain _______________ from biological samples for applications such as ________, ________________ and ________________.

RNA extraction kits, Streamlined protocols, sample types, formats, throughputs, manual, automated

______________ ensure high yields of high-quality RNA free of contaminants and inhibitors. _____________ with optimized RNA extraction reagents simplify handling and are optimized for your specific _____________, _______ and _______, as well as for __________ and ___________ processing.

GuSCN, sodium acetate, phenol, chloroform, centrifugation

The basic principle of the method is the separation of RNA from DNA and proteins after extraction with an acidic solution, which consists mainly of ______, _______, _______, and _________, followed by _________.

break down the cells or tissue

Homogenization:
(1) The first step in RNA extraction is to ________________ so that the nucleic acids are released from the cells

mortal and pestle, repetitive pipetting (resuspend)

Homogenization:
(2) Homogenization
Tissue: ___________________
Cells: _________________________

powerful chaotropic salt

Homogenization:
(3) RNA extraction methods use a ____________ solution. This ‘lysis solution’ rapidly disrupts cells without destroying their nucleic acids.

Total RNA isolation reagent, phenol, guanidine thiocyanate

Homogenization:
(4) ____________________ (TRI reagent) as ‘lysis solution’ combines ________ and ________________.

guanidinium thiocyanatephenol-chloroform, TRIreagent (or TRIzol), guanidinium isothiocyanate, protein denaturant, phenol

A common method used to isolate total RNA is _______________________ extraction. This method utilizes a chemical called ________________, which contains _____________________, a powerful ____________, and ______, an organic solvent.

RNA integrity, Piotr Chomczynski, Nicoletta Sacchi, 1987, 2006, RNAeasy (Qiagen)

The TRIreagent buffer maintains the _________, disrupts cells and denatures proteins. This method is based on the research of ___________ and ____________ in _____ & ______. It takes slightly more time than column-based methods like __________, but it can handle larger amounts of tissue or cells and therefore yields more RNA.

Detergent

Homogenization: Lysis Solution
Breaks down the hydrophobic membranes that surround cells and some cellular organelles.

Reductant

Homogenization: Lysis Solution
Inactivates Rnases.

Reductant

Homogenization: Lysis Solution
Preserves RNA during the tissue disruption process.

Chaotrope

Homogenization: Lysis Solution
Unfolds proteins and other biochemicals.

5 min, RT

Phase Separation
Homogenate (from homogenization step) must be stored for _____ at _____ to permit the complete dissociation of nucleoprotein complexes.

aqueous phase, interphase, organic phase, RNA, DNA, protein, Chloroform, hydrophobic

Phase Separation
Chloroform used to:

• Separate solution in ________, _________and ____________

• _______ in aqueous phase, _______ (interphase) and _______(organic phase)

• ________ is organic solvent. So, lysed cell components that are _________ will be trapped in these solvent (eg: membrane lipids, hydrophobic polypeptide sequences (protein) or polysaccharides etc.)

density centrifugation

Phase Separation
RNA, DNA and protein separation are based on _______________.

cold, 2-8C, DNA, aqueous

Phase Separation
Centrifugation in _______ temperature (________). If performed at high temperature, a residual amount of _______may mix in the ________ phase.

RNA precipitation

is an easy and cost-effective method for the concentration of RNA, leaving a pellet that can be resuspended in the buffer of choice.

RNA precipitate

(often visible before centrifugation) forms a gel-like or white-pellet on the side and bottom of the tube

Isopropanol

precipitates RNA from the aqueous phase.

Lithium chloride

does not precipitate tRNA and may not work as well for RNAs under ~100 nucleotides or very dilute RNAs. Lithium chloride precipitation removes nucleotides and most proteins.

75% ethanol

Wash RNA pellet away from any salt residual.

RNase free water

Clean RNA resuspended (RNase free water) to ensure stability and long-term storage.

microspectrophotometer

RNA Quality Control can be assessed by:

260, 280, 1.8-2.0, protein, solvent, Agilent Bioanalyzer, RNA integrity number

RNA Quality Control

• Ratio of the absorbance at ____ and ______ nm is used to assess the RNA purity.

• Value _________ indicates that the RNA is pure.

• If <1.8 : ______ contamination

• >2.0: ________ contamination

• The quality of RNA can be estimated by____________.

• The integrity of total RNA samples can be determined by ______________.

DNase treatment, residual genomic DNA, highly pure RNA, column purification

Troubleshooting
If you get bad purity of RNA

• Eliminating DNA contamination with _________

• To remove _______________ and obtain ______________.

• Purify with _________________.

230 nm

Wavelength when you measure the chemicals remaining in the sample

260 nm

Wavelength when you measure the nucleic acids (RNA and DNA)

280 nm

Wavelength when you measure the proteins.

1.8-2.1

A 260/280 ratio should be between ________.

Microspectrophotometer, purity, quantity, 260, RNA, DNA, 260, lower RNA, DNA, 280, proteins, purity

The ___________ measures both the ____ and ______ of the nucleic acids. The higher the value at _____ nm, the ______ the concentrations of _____ and _____. The lower the value at _____ nm, the ____ the concentrations of _____ and ______. The higher the value at _______ nm, the higher the concentrations of _________. Meaning, the ____ of the nucleic acids is very low.

OD values, nucleic acid degradation, Gel Electrophoresis

But the _______ cannot tell us the _________. To check the integrity, we need to use other techniques such at _____________.

separation, magnetic beads

There are various methods that can be used to purify mRNA from the total RNA in an extract. For example, using _________ (similar to the DNA extraction method) or using _____________.

selectively, mRNA, polyA tail

The magnetic beads with oligodT attached will bind _______ to the ______ molecules since mRNAs typically include a _________. When placed in the magnetic rack the beads containing attached mRNA will be separated from the solution. We can then pipette the solution containing all the unwanted materials; and wash the beads using 80% ethanol.

elute

After several rounds of washing, we can _____ (separate) the mRNA molecules from the magnetic beads and transfer them to a new tube.

but only a certain set of genes is expressed at a given time

Every cell contains the entire genome (the full genetic blueprint), _______________________________ The set of genes that is expressed gives a cell its characteristics, its functionalities and when applicable, its specialization. This is a dynamic process, with genes expression being turned on and off in response to internal and external stimuli.

transcribed, RNA molecule, messenger RNA, regulatory RNAs, structural RNAs

A gene is considered expressed when it is ________ and a functional product produced. The direct product of transcription is an __________: _______ (mRNA), ____________ (such as miRNAs) or ____________ (like rRNA).

Next Generation Sequencing, Quantitative PCR

To measure gene expression levels, different techniques can be used, for example ______________ or ____________________.

physiological state

Why resort to RNA studies if DNA information is available and is similar across the cells of an organisms?

(1) The genes that are expressed and the level they are expressed are different depending on the _____________ of the person.

proteins, functional RNA species

Why resort to RNA studies if DNA information is available and is similar across the cells of an organisms?

(2) Instead of dealing only with DNA, it is also necessary to work with gene expression products such as the _________ produced and the _________________ on its precursor mRNA.

molecular level

Why resort to RNA studies if DNA information is available and is similar across the cells of an organisms?

(3) mRNA expression may show the overall cellular activity at the ____________, and thus, the molecular regulatory mechanisms of certain physiologic conditions like diseases may be explored, and novel biomarkers may be identified.

tularemia, malaria, dengue, breast cancer, tubeculosis

Why resort to RNA studies if DNA information is available and is similar across the cells of an organisms?

(4) From gene expression studies, effective clinical diagnostic tests may be developed such as in the case of _______, uncomplicated _____, ______, _______________, and _____________ among the others.

Obesity, genes

__________ has become a worldwide epidemic, and dramatic changes in lifestyle (increased caloric intake and decreased exercise) are without a doubt the most important reason. However, during the past few decades it has become evident that _____ also influence obesity status, and increasing effort is being made to identify genes and genetic variants of importance to obesity.

mutation, expression level of genes

A _______ in a gene/genetic element (eg transcription factor, regulatory factor) may alter the gene’s original function, causing an increased tendency for obesity. Also an alteration in the ____________ may affect the obesity status.

high, expression level

A ____ mRNA level reflects a high __________.