Nucleic Acid (NA) Isolation, Quantification & Quality assessment

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Nucleic Acid (NA) isolation is a key step in many molecular biology workflows impacting downstream steps especially enzymatic reactions. Commonly used biological samples used for life sciences are as tabulated below:

Clinical and HLA	Applied Markets	Basic Research
Blood, tissue biopsies, Circulating tumor cells (CTC’s), Stem cells, Cultured cells, Urine, feces, buccal swabs, sputum, hair follicles, Formalin Fixed Paraffin embedded (FFPE) samples	Cultured cells, environmental samples-soil, bio-waste, food samples, forensic samples, animal tissues	Tissues-Plants or animals, culture cells, Single cell analysis, blood, bacterial cultures

Solutions from

The challenges posed by each of the samples w.r.t NA isolation is unique. While environmental samples require steps to remove inhibitors, clinical samples such as urine require additional concentration steps to collect NA in small volumes and forensic crime scene samples could be limiting in quantity and subject to harsh environments degrading the nucleic acid in the sample.

DNA
RNA
Common Equipment

Sample collection Homogenization DNA isolation DNA quantitation & quality assessment DNA storage

Sample collection

Sample collection incorporating good laboratory practices include:

Use of nuclease free tubes, tips, buffers and water
Cleaning surfaces using DNA away to eliminate DNA exonuclease and endonuclease contamination
Use of appropriate gloves.
Protection of surfaces using bench protectors.

Clinical samples and those suspected to contain infectious material should be handled with the relevant Biosafety precautions- biosafety cabinets, hoods, appropriate clothing & gloves and other handling guidelines.

Homogenization

Adequate disruption of the cellular or tissue architecture to release the nucleic acids in the cell can affect the quality and quantity of Nucleic acid isolated. Hence, the homogenization step in the protocol has to be optimized based on sample type and source. Several tools and options are available to achieve a homogeneous cell mixture suitable for downstream Nucleic acid (NA) isolation. The following table captures most often deployed solutions:

Sample type	Sample properties	Homogenization & other solutions	Procedure Modifications
Animal Tissues	Hard tissues (bones, teeth), nuclease-rich (spleen, liver), fatty tissues (brain)	TH Tissue Homogenizer Tissue Master THQ Digital Tissue homogenizer Hard Tissue OMNI Tip Soft Tissue OMNI Tip Omni General Laboratory Homogenizer (GLH 850)	Thorough disruption with appropriate tissue homogenizer and tips and ensure complete mixing with lysis buffer from DNA/RNA isolation kits Alternate Freeze tissue and grinding
Food & Plant	Hard grains, emulsions, large volumes, Fibrous and difficult tissues to disrupt	Omni Prep homogenizer with Omni Hard Tissue Tips Omni Mixer Omni General Laboratory Homogenizer (GLH 850)	Optimize homogenization, collect debris by centrifugation and re-homogenize
Environmental	Bacterial sample cell wall lysis critical to release nucleic acids Nanoparticle dispersion	Omni Ruptor 4000 Ultrasonic Homogenizer Omni Sonic Ruptor 400 Homogenizer Bead Ruptor Micro-Organism Lysing Mix (2 mL Tubes) Nuclease Free- 50 Pack Bead Mill Bacterial DNA Purification Kit – 50 Prep	Ensure thorough disruption, for bacteria- centrifuge and re-sonicate or homogenize bacterial pellet.
Soil Samples		Bead Ruptor Micro-Organism Lysing Mix (2 mL Tubes) Nuclease Free- 50 Pack Soil DNA isolation kit
Cellular	From blood, cell cultures, phage particles	Bead Ruptor Omni Mixer Omni Ruptor 4000 Ultrasonic Homogenizer	Ensure complete mixing with lysis solution after homogenization

DNA isolation

DNA isolation or genomic DNA (gDNA) isolation protocols aims to dissociate DNA from the cellular protein machinery and release into solution. Most commonly used solutions for DNA isolation employ chaotropic salts with or without organic components (phenol) to lyse the cell. This step prevents DNA degradation by exo or endonucleases in the cell. Subsequently, some kind of separation- either on a column, by centrifugation or magnetic bead based methodology is used to capture the pure DNA.

Special kits for DNA isolation from tissues, plants, forensic and microbial samples are:

Tissues, Cells & Blood
Plants
Forensic samples
Food & Microbial samples

Automation using KingFisher instrument and solutions is available for processing 24 to 96 samples or higher in a single run.

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Plant RNA isolation aid

Automation solutions for DNA isolation

MagMAX™ Express Magnetic Particle Processor
MagMAX™ Express-96 Deep Well Magnetic Particle Processor
MagMAX™ Express-96 Standard Magnetic Particle Processor
Learn more about MagMAX instruments

Kingfisher Presto & Hamilton Microlab Nimbus Hd

Microlab VANTAGE Liquid Handling System

ChemagicSTAR

Genomic DNA from Tissues, Cells and Blood

	DNAzol Reagent for whole blood	DNAzol™ Reagent for solid and liquid samples	PureLink ™ Genomic DNA Mini Kit	MagMAX™ DNA Multi-Sample Ultra Kit
Catalog Number	10974020	10503027	K182001	A25920
Isolation technology	Uses novel organic & guanidine-detergent lysing solution selectively allows DNA precipitation while lysing RNA. (Manual- not for high throughput)	Uses novel organic & guanidine-detergent lysing solution selectively allows DNA precipitation from cell lysate. (Manual- Not for high throughput)	Silica spin column methodology (Manual- not for high throughput)	Magnetic bead–based purification scalable from processing 12 to 500 samples a day. Automated Protocols, High Throughput-Compatible, Manual Protocols
Prep time	30 minutes	10-30 minutes	20-30 minutes	~45 minutes
Sample Types	Whole blood, serum samples for viral DNA	Cell, whole blood or tissues	Blood, Tissues, cells, Bacteria, swabs, and blood spots, with a familiar	Whole blood, buccal cells, saliva, urine, blood cards, mouth rinse, and tissue
Starting Material	1 ml of DNAzol® Reagent for genomic DNA from 0.5 ml of whole blood to yield 10-20 µg DNA. Can be used for isolation of apoptotic fragments from whole blood and viral DNA from serum	1 mL of DNAzol® Reagent can be used to isolate genomic DNA from 1–3 x 107 cells, from 0.1 mL of whole blood, or from 25–50 mg tissue.	5 x 10^6 to 1 x 10^8 cells, Up to 200 mg tissue	1 buccal swab Whole blood- 50 to 350 µL Urine (not conc)- up to 400 µL Saliva up to 150 µL Mouth Rinse ( 1-5 mL ) Blood spot cards Mammalian tissue (up to 10 mg)
Applications	Southern blotting, cloning, PCR, restriction endonuclease digestion, and dot blot hybridization.	Southern blotting, cloning, PCR, restriction endonuclease digestion, and dot blot hybridization.	Genotyping, PCR, Real-Time Quantitative PCR (qPCR), Sequencing, Southern Blotting	Next-Generation Sequencing, Real-Time Quantitative PCR (qPCR)

Genomic DNA from Plants

	ChargeSwitch™ gDNA Plant Kit	MagMAX™ Plant DNA Isolation Kit	Plant DNAzol Reagent
Catalog Number	CS1800010	A32549-96 preps A32580-384 preps	10978021
Isolation technology	Magnetic Bead based extraction. Pure DNA with no guanidine or organic contamination. High Throughput-Compatible	Magnetic bead technology, eliminates the need for phenol/chloroform extraction & alcohol precipitation. High throughput-Compatible	Novel organic & guanidine-detergent lysing solution for selective DNA precipitation and RNA lysis. Not High Throughput-Compatible (Manual)
Prep time	15 minutes	30 minutes	60-70 minutes
Sample Types	Fungal and plant samples, including leaves, tissues and seeds	Suitable for most plant types and parts, including woody, polyphenol-rich, and lignified samples	Plant tissues such as leaf, seed stem, root, and callus
Starting Material	50-100 mg of fungi and plant samples- including leaves, tissues & seeds to yield up to 7 µg of gDNA	10–100 mg plant samples	Up to 100 mg tissue, Up to 5×106 cells
Applications	Genetically modified organism (GMO) screening, PCR, restriction enzyme digestion, and Southern blotting.	PCR, Real-Time PCR (qPCR) Next-generation sequencing (NGS)	Cloning, PCR, Sequencing, Southern Blotting

Genomic DNA from Forensic samples

	AutoMate Express™ Forensic DNA Extraction System	MagMAX™-96 DNA Multi-Sample Kit	ChargeSwitch® Forensic DNA Purification Kit	iPrep™ ChargeSwitch™ Forensic Kit
Catalog Number	4441763	4413022	CS1120010	IS10002
Isolation technology	Bench-Top instrument which utilizes PrepFiler Express™ and PrepFiler Express BTA™ chemistries packaged in pre-filled, foil sealed cartridges. Protocol card pre-programmed for purifying nucleic acids from forensic samples.	Magnetic bead-based nucleic acid isolation technology free from inhibitors which affect downstream reactions: genotyping assays, CE and next-generation sequencing, and high resolution melting analysis	Magnetic bead-based technology to isolate genomic DNA without the need for hazardous chemicals, centrifugation, or vacuum manifolds (Ideal for automation and high throughput applications)	ChargeSwitch® technology is based on an unique, ionizable nucleic acid-binding ligand whose charge can be switched based on the pH of the surrounding medium. Purification avoids the use of guanidine, ethanol, and other reagents. Not High Throughput-Compatible (Manual)
Prep time	30 minutes (up to 13 samples)	20 minutes	20-30 minutes	20 minutes
Sample Types	Challenging samples such as bones, teeth and adhesive based samples such as cigarette butts and tape lifts	Blood and blood cards, tissue buccal swabs Buffy coats, and cultured cells. Ideal for samples containing enzymatic inhibitors matrix (Haeme, Heparin, salts, protein) and removal of inhibitors from the purification method itself (phenol, salts, alcohol)	Suited for environmentally degraded samples Dried blood spots on paper, clothing Hair follicles and hair shafts Cigarette butts Cigarettepaper Envelopes Drinking vessel swabs Chewing gum Sperm head cells Vaginal epithelial cells Door handle swabs Strip removed cells (e.g. hats, coats, and gloves) “Touch DNA” (e.g. tools, mobile phones, and microscopes) Dyed denim	Dried blood spots on paper and clothing Hair follicles and hair shafts · Cigarette butts Semen “Touch DNA” (e.g. tools, mobile phones, and microscopes)
Applications	For Research, Forensic or paternity genotyping tests analyzing short tandem repeats (STRs)	Genotyping, PCR, Real-Time Quantitative PCR (qPCR), Sequencing	Forensic Crime scene sample types for STR profiling, PCR	Cloning, Forensic Testing, PCR, Sequencing, Southern Blotting

Genomic DNA for Food Safety and Microbial Testing applications

	PrepSEQ™ Nucleic Acid Extraction Kit for Food and Environmental Testing	PureLink™ Pro Quick96 Plasmid Purification Kit * non-genomic (plasmid DNA) prep	MagMAX™ Total Nucleic Acid Isolation Kit
Catalog Number	4480466	K211024A	AM1840
Isolation technology	Enzymatic & magnetic beads. Automated Protocols, High Throughput-Compatible. Ideal with MagMAX™ Express-96 Deep Well Magnetic Particle Processor	Silica plate extraction chemistry with 96-well plate design for manual or automated processing of plasmid DNA. Automated Protocols, High Throughput-Compatible	Zirconia beads for the mechanical disruption of difficult pathogens. DNA isolation using magnetic bead technology.
Prep time	6-20 hrs (based on Microbial enrichment step)	<45 minutes	~45 minutes
Sample Types	Bacteria, Food & Environmental Samples, Liquid Samples (e.g. Serum)	E. coli strains grown in up to 5 ml LB, 3 ml 2xYT, or 3 ml TB	Bacterial and cell culture samples
Starting Material	100 mg of tissue or 107 cells (requires 1 mL reagent)	1.2-1.5 ml lysate⁄well yielding up to 15ug plasmid DNA	175 µl liquid samples of bacterial cultures
Applications	PCR, Real-Time Quantitative PCR (qPCR), Ideal for Listeria, E.coli, Salmonella and other bacteria testing in Food and environmental samples	Cloning, Next-Generation Sequencing, PCR, Sequencing, Restriction Enzyme digestion.	Veterinary use for PCR, Real-Time PCR (qPCR)

Other kits: PureLink™ Microbiome DNA Purification Kit (Invitrogen™)
Vacuum manifold for plasmid purifications: FastVac™ Vacuum Manifold

DNA quantitation & quality assessment

Assessment of nucleic acid concentration can be done via spectrophotometric or fluorescence based measurements. Spectrophotometric absorbance measurements at 260 nm and 280 nm, provide an estimate of the amount of the DNA or RNA and protein in the sample. Alternate measurements at 230 nm allow evaluation of contaminants from the NA isolation protocol such as phenol. Various applications require different concentrations of sample and purity. Ratios of A260/A280 nm of 1.7 or higher indicate high quality nucleic acid ideal for most applications.

Fluorescence measurements with dyes that specifically bind dsDNA or RNA are recommended for applications such as Next Generation Sequencing, Microarrays and high throughput Real-time PCR (qPCR). Specialized fluorimeters such as the Qubit 2.0 or 3.0 systems with dedicated kits allow NA measurements for such applications. For more on comparison between Absorbance vs. Fluorescence see below and read more at:

UV measurement guidelines for RNA analysis

Absorbance	Indicates presence of	Target ratios
230 nm	Organic compounds, sugars, urea, salts	A260/A230 > 1.8
260 nm	All nucleic acids	A260 ≈ 0.1–1.0
270 nm	Phenol	A260/ A 270 > 1.0
280 nm	Protein	RNA: A260/A280 ≈ 2.0 DNA: A260/A280 ≈ 1.8
330 nm	Light scattering	A330 = 0

Absorbance Measurements	Fluorescence Measurements
Cannot discriminate between DNA and RNA if both present in the sample	Quantitation done by binding of specific dyes unique for dsDNA, RNA, small RNA- measures exact level of each species
Not ideal for very limited sample concentrations 10ng/µL and lower	Ideal for very limited sample concentrations 1 ng/µL or lower

Kits	Sample starting concentration range for kit
Qubit ds DNA HS assay	10pg/µL-100ng/µL
Qubit sdDNA BR assay	100pg/µL-1µg/µL
Qubit ssDNA assay	50pg/µL-200 ng/µL
Qubit RNA assay	250 pg/µL-100ng/µL
Qubit RNA BR assay	1ng/µL-1µg/µL
Qubit protein assay	12.5µg/mL-5mg/µL

Ensure optimal RNase I treatment of DNA samples. To check, run on an 1% agarose with appropriate size standards to visualize high molecular weight DNA and ensure RNA (smear at low molecular weights) is removed

DNA storage

High mol. weight genomic DNA

Storage of purified DNA after isolation can be carried out based on long term or short term usage. Ideally, DNA should be stored in buffered solutions such as Tris EDTA (TE) at pH 8.0. A small aliquot of DNA can be stored at 4°C refrigerators for repeated uses. For long term storage, aliquot and freeze in -80°C freezers. Frozen samples should not undergo repeated freezing and thawing as this degrades large molecular weight genomic DNA.

Typical steps in a manual RNA isolation

Sample collection, stabilization and storage RNA isolation RNA quality and quantification RNA storage

Sample collection, stabilization and storage

Working with RNA poses its own set of challenges. RNA is required for studies involving Gene Expression, Transcriptome analysis, small RNA and microRNA analysis (miRNA), RNA interference (RNAi) , structural RNA studies or RNA virus detection.

Unlike its deoxy counterpart, the ribonucleic sugar in RNA is susceptible to alkaline hydrolysis. Once released out of the cell, the RNA is prone to degradation by Ribonucleases (RNAses) which are ubiquitous in the sample and surroundings.

IGB Tips: 4 R’s for good quality RNA!

RNAase–free sample collection using Nuclease free tips, tubes, buffers & decontaminate surfaces with RNAzap
Resuspend samples immediately in RNAlater™ Stabilization Solution
Research ideal combination of isolation protocols: Combinations suited for all sample in the experiment (Guide )
Recovered RNA should be stored in The RNA storage or RNAsecure™ Resuspension Solution

If the experiment involves work with infectious agents additional protection will need to be deployed as described below:
Protection of surfaces using bench protectors. Clinical samples and those suspected to contain infectious material should be handled with the relevant Biosafety precautions- biosafety cabinets, hoods, appropriate clothing & gloves and other handling guidelines. Visit for more biosafety solutions.
The Oragene•RNA for Expression Analysis Self-Collection Kit is the only all-in-one system for the collection, stabilization and transportation of high quality mRNA from saliva.

RNA isolation

Protocol modifications to improve RNA yield and quality

Tissue Characteristics	Symptoms	Representative Tissues	Procedure Modifications
Fibrous Tissues (put pictures here)	Difficult to homogenize	Heart muscle Skeletal muscle	Thorough disruption with appropriate tissue homogenizer/ Freeze tissue and grind
Nucleic Acid-rich	Viscous lysate, incomplete phase separation, precipitation on addition of isopropanol	Spleen Thymus	Dilute lysate Do additional extraction
Protein and Lipid-rich	White flocculent material in aqueous phase of extraction	Brain Plant Tissue	Dilute lysate Modify extraction(more CHCI3)
Nuclease-rich	Degraded RNA	Spleen Pancreas Thymus	Efficient disruption Freeze tissues and grind

Guide & Brochure: Match our RNA isolation solution to your experimental needs!

RNA isolation from tissues, cells and blood

	TRIzol Reagents	Trizol Plus Reagents	PureLink Mini Kits	MagMAX Kits
Catalog Number	15596026	12183555	12183020 (other larger size kits available)	AM1840
Isolation technology	Organic phenol extraction and alcohol precipitation. Manual- Not for high throughput	Dual purification using Organic extraction and spin columns. (Manual- Not for high throughput)	Silica spin column based methodology (Manual- not for high throughput)	Scalable, flexible format with magnetic beads (Ideal for high throughput applications)
Prep time	~1 hour	20 minutes	< 20 minutes	~45 minutes
Sample Types	Bacteria, Blood, Cells, Plant Samples, Tissue, Viral Samples, yeast. Ideally suited for samples that are difficult to lyse	Bacteria, Cells, Plant Samples, Tissue, Yeast *Highly recommended for fatty and nuclease rich samples	Bacteria, Blood, Cells, Plant Samples, Tissue, Yeast, *specific formulation kits available for blood, virus, miRNA	Kits for cell-free, Tissues, cells, stabilized blood, serum/plasma, urine, plants, viral, pathogen extraction and FFPE samples
Starting Material	100 mg of tissue or 10⁷ cells (requires 1 mL reagent)	Up to 200 mg tissue, Up to 5 x 10⁸ cells	5 x 10^6 to 1 x 10^8 cells, Up to 200 mg tissue	Up to 100 mg tissue, Up to 5×10⁶ cells
Applications	Cloning, Northern Blotting, Nuclease Protection Assays, Real-Time Quantitative PCR (qPCR), Reverse Transcriptase PCR (RT-PCR), cDNA Library Construction	Microarray Analysis, Northern Blotting, Nuclease Protection Assays, Nucleic Acid Labeling, Real-Time Quantitative PCR (qPCR), Reverse Transcriptase PCR (RT-PCR), cDNA Library Construction	Microarray Analysis, Next-Generation Sequencing, Northern Blotting, Nuclease Protection Assays, Real-Time Quantitative PCR (qPCR), Reverse Transcriptase PCR (RT-PCR), cDNA Library Construction	Northern Blotting, PCR, Real-Time Quantitative PCR (qPCR), Reverse Transcriptase PCR (RT-PCR), Southern Blotting
Types of RNA isolated	Total RNA, Transcriptome RNA, micro RNA	Total RNA (yield up to 1000ug)	Total RNA	Total RNA, microRNA

Microbial RNA or RNA & DNA isolation solutions

	RiboPure™ RNA Purification Kit, bacteria	MICROBEnrich Kit	MagMAX™ Pathogen RNA/DNA Kit	PureLink® Viral RNA/DNA Mini Kit
Catalog Number	AM1925	AM1901	4462359	12280050
Isolation technology	Dual organic and Silica column based method for isolation of RNA. Manual (not for high throughput applications)	Magnetic bead based hybridization capture. Enriches for microbial mRNA in a host-microbe mixed RNA sample by selective depletion of host mRNA and rRNA. Host: Human, mouse, rat	Isolation of RNA and DNA from viruses and G- bacteria. Kit based on magnetic particle based 96-well format. Hi-throughput instruments: KingFisher™ Flex Magnetic Particle Processor, MagMAX™ Express Magnetic Particle Processor, MagMAX™ Express-96 Deep Well Magnetic Particle Processor, MagMAX™ Express-96 Standard Magnetic Particle Processor	The kit is designed for isolation of viral RNA or DNA in a low volume. It is a silica spin column based isolation resulting in NA free of proteins and nucleases. The final NA is eluted in 10-50uL elution volumes typically used for viral detection and genotyping.
Prep time	About 45 mins		45 minutes	<45 minutes
Sample Types	Ideal for difficult to lyse G+ bacterial samples	Host-microbe mix total RNA	Serum and plasma; nasal, tracheal, and cloacal swabs; ear notches) • Whole blood • Semen • Oral fluid • Feces • Biomed Diagnostics InPouch™ TF (Tritrichomonas foetus) culture	Cell-Free Fluids, Cerebrospinal Fluid, Plasma, Serum
Starting Material	Up to 10^9 cells	20 reactions x 25 µg RNA total RNA per reaction	Variable	Viral RNA and DNA from ≤500 µL cell-free samples
Applications	RNA sequencing, microarray analysis, real-time PCR (qPCR), reverse transcription qPCR (RT-qPCR), northern blotting, and cDNA library construction	Microarray analysis	PCR, Real-Time Quantitative PCR (qPCR), Reverse Transcriptase PCR (RT-PCR)	Northern Blotting, Real-Time Quantitative PCR (qPCR), Reverse Transcriptase PCR (RT-PCR)
Type of RNA isolated	Total RNA	Total RNA	Total RNA	Total RNA

Automation solutions for RNA isolation

MagMAX™ Express Magnetic Particle Processor
MagMAX™ Express-96 Deep Well Magnetic Particle Processor
MagMAX™ Express-96 Standard Magnetic Particle Processor
Learn more about MagMAX instruments

Kingfisher Presto & Hamilton Microlab Nimbus Hd

Microlab VANTAGE Liquid Handling System

ChemagicSTAR

RNA quality and quantification

UV measurement guidelines for RNA analysis

Absorbance	Indicates presence of:	Target ratios
230 nm	Organic compounds, sugars, urea, salts	A260/A230 > 1.8
260 nm	All nucleic acids	A260 ≈ 0.1–1.0
270 nm	Phenol	A260/ A 270 > 1.0
280 nm	Protein	RNA: A260/A280 ≈ 2.0 DNA: A260/A280 ≈ 1.8
330 nm	Light scattering	A330 = 0

Absorbance Measurements	Fluorescence Measurements
Cannot discriminate between DNA and RNA if both present in the sample	Quantitation done by binding of specific dyes unique for dsDNA, RNA, small RNA- measures exact level of each species
Not ideal for very limited sample concentrations 10ng/µL and lower	Ideal for very limited sample concentrations 1 ng/µL or lower

Kits	Sample starting concentration range for kit
Qubit ds DNA HS assay	10pg/µL-100ng/µL
Qubit sdDNA BR assay	100pg/µL-1µg/µL
Qubit ssDNA assay	50pg/µL-200 ng/µL
Qubit RNA assay	250 pg/µL-100ng/µL
Qubit RNA BR assay	1ng/µL-1µg/µL
Qubit protein assay	12.5µg/mL-5mg/µL

Ensure optimal DNase I treatment of RNA samples. To check run on 1% agarose with appropriate size standards to ensure no high molecular weight bands corresponding to gDNA. Additional QC checks with Qubit assays can help measure concentration of RNA in the final isolate.

RNA storage

Purified RNA should be stored in THE RNA storage or RNA secure™ Resuspension Solution.
Ideally, RNA should be stored in at neutral or slightly acidic conditions. A small aliquot of RNA can be stored at 4°C Refrigerators for repeated uses. For long term storage, aliquot and freeze in -80°C Freezers. Frozen samples should not undergo repeated freezing and thawing as RNA is very susceptible to degradation.